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WO2025122569A1 - Récepteur de lymphocytes t spécifique du néoantigène kmt2a::aff1 et son utilisation en immunothérapie adoptive - Google Patents

Récepteur de lymphocytes t spécifique du néoantigène kmt2a::aff1 et son utilisation en immunothérapie adoptive Download PDF

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WO2025122569A1
WO2025122569A1 PCT/US2024/058390 US2024058390W WO2025122569A1 WO 2025122569 A1 WO2025122569 A1 WO 2025122569A1 US 2024058390 W US2024058390 W US 2024058390W WO 2025122569 A1 WO2025122569 A1 WO 2025122569A1
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cell
cells
recombinant
host cell
tcr
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Paul G. Thomas
Ricky TIRTAKUSUMA
Mohamed A. GHONIM
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St Jude Childrens Research Hospital
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St Jude Childrens Research Hospital
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    • 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
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Fusion-driven leukemias can be clinically aggressive and associated with poor prognosis.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • MPAL mixed phenotypic leukemia
  • some subtypes characterized by specific genetic fusions continue to be associated with poor outcome.
  • leukemias harboring certain KMT2A rearrangements can have a dismal prognosis, with a 5-year event free survival less than 30%.
  • agents that target fusion- associated pathways are currently being tested, including Menin and NSD1 inhibitors.
  • chimeric antigen receptor T cells have been practice-changing in B-cell malignancies and have shown some promise in AML (Vishwasrao et al. (2022) Cancers 14(5):1241), but do not target fusion- specific antigens.
  • T-cell receptor (TCR)-engineered T cells and cancer vaccines have also shown potential.
  • WT1 and minor histocompatibility antigens have been major candidates of these novel therapeutics (Maslak et al. (2016) Blood Adv. 2 (3):224-234; Kreutmair et al. (2022) Cancer Immunol. Immunother. 71(12):2913-2928; Tawara et al. (2017) Blood 130 (18):1985-1994; Chapuis et al. (2019) Nat. Med. 25(7):1064- 1072; Dossa et al. (2018) Blood 131(1):108-120) in treating AML.
  • RNA-interference molecules and other inhibitors targeting different fusions including BCR::ABL1 (Wilda et al. (2002) Oncogene 21(37):5716- 5724; Thiesing et al. (2000) Blood 96 (9):3195-3199), KMT2A: :AFF1 (Thomas et al. (2005) Blood 106(10):3559-3566), KMT2A: :MLLT1 (Horton et al.
  • This invention provides a recombinant T cell receptor (TCR), or antigen binding fragment thereof, including an a- chain variable domain having a complementarity determining region 3 (CDR3) amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and/or a ⁇ -chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID N0:2).
  • TCR T cell receptor
  • Isolated polynucleotides encoding the recombinant TCR and an expression vector and recombinant host cell including the polynucleotides operably linked to an expression control sequence are also provided.
  • the invention further provides an adoptive immunotherapy method for treating a subject diagnosed with, suspected of having, at risk for developing, or at risk for recurrence of a leukemia that expresses a KMT2A::AFF1 fusion neoantigen by administering to the subject a recombinant host described herein.
  • a unit dose form comprising a recombinant host cell as described herein is also provided
  • FIG. 1. shows lysis of autologous blasts by expanded T cells from samples SJINF002, PDX SJALL016500, and SJALL048457 after an 18-hour incubation, as measured by flow cytometry.
  • FIG. 2 shows lysis of autologous blasts by expanded T cells from AML samples SJAML001441 (assessment on PDX blasts), SJAML043616, SJAML031669, SJAML005142, and SJMLL012 and from MPAL samples SJMPAL016107 and SJMPAL012424 after 18-hour incubations, as measured by flow cytometry.
  • FIGS. 3A-3E show co-culture of TCRs that were reactive to leukemia blasts and fusion genes. The reactive TCRs included TCR SJINF002_2 ("_2" indicates ranked 2 by frequency) (FIG. 3A), TCR SJINF013_2 (FIG.
  • TCR SJAML00144_2 (FIG. 3C)
  • TCR SJAML030459_3 (FIG. 3D)
  • SJAML030459_6 (FIG. 3E).
  • the reactivity was measured by IFN- ⁇ ELISPOT (left axis) and flow cytometry upregulation of 0X40 or 4-1BB (right axis).
  • the fusion genes were KMT2A exon 10-AFF1 exon 4 (SJINF002), KMT2A exon 10-AFF1 exon 5 (SJINF013), NUP98 exon 11-NSD1 exon 6 (SJAML001441), PICALM exon 19-MLLT10 exon 4(SJAML030459), RUNX1 exon 6-RUNX1T1 exon 2 (SJAML030471).
  • FIG. 4 shows reactivity of TCRs SJINF002_2, SJAML001441_1, SJAML001441_2, SJAML0304593, and SJAML030469__6 when co-cultured with leukemia blasts for 18 hours. Blast lysis was measured by flow cytometry. TCR clonotype was listed above the TCR bar.
  • FIGS. 5A-5C show reactivity of TCRs SJINF002 clone 2 (FIG. 5A) and TCR clones 3 and 6 in sample SJAML030459 (FIG. 5B-5C, respectively) co-cultured with 293T CIITA or K562 cells expressing single HLA alleles of the corresponding patients as antigen-presenting cells (ABCs).
  • the RNA-fusion gene was overexpressed on the APCs.
  • the reactivity was assessed by flow cytometry assessment of 0X40 and 4-1BB upregulation or by IFN- y ELISPOT.
  • FIG. 6 shows reactivity of T cells expressing TCR SJINF002__2 with APCs HLA-DPAl*02:01/DPBl*01:01 pulsed with 1 mg/mL RIRVDFKQTYSNEVH peptide.
  • the cells were washed, co- cultured and reactivity was measured by flow cytometry of CD4 + 0X40 upregulation (left panel) and IFN- ⁇ secretion (right panel) .
  • FIGS. 7A-7D show that TCR SJINF002_2-expressing T cells reduce tumor burden and improve survival.
  • FIG. 7A % leukemia blast SJINF002 engraftment at days 8, 22 and 36 post- T cell transfer.
  • FIG. 7B expansion of the SJINF0022 TCR T cells at days 8, 22 and 36 post-T cell transfer.
  • FIG. 7C % changes in body weight over time post-T cell transfer.
  • FIG. 7D % survival after T cell injection.
  • Fusion gene-derived neoantigen KMT2A::AFF1 is the most common mutation in infant leukemia and independently defines a poor prognosis. The 5-year event-free survival is less than 30%, suggesting the urgency of improved therapy modality.
  • This invention relates to a specific TCR that has been identified as recognizing the KMT2A::AFF1 fusion neoantigen presented by HLA-DPAl*02:01_DPBl*01:01.
  • the data presented herein show that the adaptive immune system targets this unique tumor protein such that the TCR specifically recognizing and reactive to the KMT2A: :AFF1 fusion neoantigen is of use in targeting and killing leukemia expressing this unique fusion neoantigen.
  • this invention provides recombinant TCR or antigen binding fragment thereof, a polynucleotide and expression vector encoding the recombinant TCR, and a recombinant host cell containing the same for use in adoptive immunotherapy methods for treating a subject diagnosed with, suspected of having or at risk for developing or recurrence of leukemia, wherein the leukemia cells express a KMT2A::AFF1 fusion neoantigen.
  • T cell receptor refers to an immunoglobulin superfamily member (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al.
  • a TCR can be found on the surface of a cell or in soluble form and generally is composed of a heterodimer having ⁇ and ⁇ chains (also known as TCR ⁇ and TCR ⁇ , respectively), or ⁇ and ⁇ chains (also known as TCR ⁇ and TCR ⁇ , respectively).
  • the extracellular portion of TCR chains (e.g., ⁇ -chain, ⁇ -chain) contain two immunoglobulin domains, a variable domain (e.g., ⁇ -chain variable domain or V ⁇ , ⁇ - chain variable domain or Vp; typically amino acids 1 to 116 based on Kabat numbering) at the N-terminus, and one constant domain (e.g., ⁇ -chain constant domain or C ⁇ , typically amino acids 117 to 259 based on Kabat, ⁇ -chain constant domain or C ⁇ , typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane.
  • a variable domain e.g., ⁇ -chain variable domain or V ⁇ , ⁇ - chain variable domain or Vp; typically amino acids 1 to 116 based on Kabat numbering
  • constant domain e.g., ⁇ -chain constant domain or C ⁇ , typically amino acids 117 to 259 based on Kabat, ⁇ -chain constant domain or C ⁇ , typically amino acids 117 to 2
  • variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al. (1990) Proc. Natl Acad. Sci. USA 87:9138,; Chothia et al. (1988) EMBO J. 7:3745).
  • CDRs complementary determining regions
  • FRs framework regions
  • a TCR is found on the surface of T cells (or T lymphocytes).
  • the source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.
  • the recombinant TCR is from a human.
  • the recombinant TCR of the invention includes an a-chain constant domain having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any value or range therebetween) to a human a-chain constant domain having an amino acid sequence as set forth in SEQ ID NO:81 or SEQ ID NO:82: [0021]
  • the recombinant TCR of the invention includes a ⁇ -chain constant domain having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any value or range therebetween) to a human ⁇ -chain constant domain having an amino acid sequence as set forth in SEQ ID NO:83 or SEQ ID NO:84 :
  • the invention provides a recombinant TCR, or antigen binding fragment thereof, that specifically binds to or is specific for a KMT2A::AFF1 fusion neoantigen.
  • a "KMT2A: :AFF1 fusion neoantigen” or “KMT2A: :AFF1 antigen” refers to the neoantigen resulting from the translocation t(4;11) (q21;q23). It accounts for approximately 5-10% of newly diagnosed cases of ALL and has been reported in biphenotypic ALL, T-ALL, and in acute myeloid leukemia usually M4 or M5 subtypes.
  • KMT2A and AFF1 are known in the art and available under GENBANK Accession Nos. NC_000011.10 (Reference GRCH38.pl4 primary assembly; range 118436492..118526832) and NC_000004.12 (Reference GRCH38.pl4 primary assembly; range 86935011. .87141039), respectively.
  • the KMT2A: :AFF1 antigen results from a breakpoint located at exons 10 and 4, respectively.
  • binding protein e.g., TCR
  • Binding proteins may be classified as “high affinity” binding proteins or binding domains or as “low affinity” binding proteins.
  • “High affinity” binding proteins refer to those binding proteins having a K a of at least about 10 7 M -1 , at least about 10 8 M" 1 , at least about 10 9 M -1 , at least about 10 10 M -1 , at least about 10 11 M -1 , at least about 10 12 M -1 or at least about 10 13 M -1 .
  • “Low affinity” binding proteins refer to those binding proteins or binding domains having a K a of up to about 10 7 M -1 , up to about 10 6 M -1 , up to about 10 5 M -1 .
  • affinity may be defined as an equilibrium dissociation constant (K d ) of a particular binding interaction with units of M (e.g., about 10 -5 M to about 10 -13 M).
  • a variety of assays are known for assessing binding affinity of a binding protein (e.g., TCR) with a particular target, as well as determining binding domain affinities, such as western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660; Wilson (2002 Science 295:2103; Wolff et al. (1993)
  • the invention provides a recombinant KMT2A::AFF1-specific TCR, or antigen binding fragment thereof, including an a-chain variable domain having a CDR3 amino acid sequence of CSVRRNSNYQLIW (SEQ ID N0:l), or a sequence sharing about 90% sequence identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).
  • the invention provides a recombinant KMT2A::AFF1-specific TCR, or antigen binding fragment thereof, including a ⁇ -chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2), or a sequence sharing about 90% sequence identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).
  • the invention provides a recombinant KMT2A: :AFF1-specific TCR, or antigen binding fragment thereof, including an a-chain variable domain having a CDR3 amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and a ⁇ -chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2).
  • the invention provides a recombinant KMT2A::AFF1-specific TCR, or antigen binding fragment thereof, including an a-chain variable domain having a CDR3 amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and/or a ⁇ -chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2) and further including an a-chain constant domain having an amino acid sequence of SEQ ID NO:81 or SEQ ID NO:82 and/or ⁇ -chain constant domain having an amino acid sequence of SEQ ID NO:83 or SEQ ID NO:84.
  • a "binding domain,” "antigen binding domain,” or “antigen binding fragment” refers to a domain or portion of a KMT2A::AFF1-specific binding protein (e.g., TCR) responsible for the specific KMT2A::AFF1 binding.
  • a KMT2A::AFF1-specific binding protein e.g., TCR
  • a KMT2A::AFFl-specific antigen binding fragment alone (i.e., without any other portion of a KMT2A::AFFl-specific binding protein) can be soluble and can bind to KMT2A::AFF1 with, e.g., a Kd of less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 M.
  • an KMT2A::AFF1 antigen binding fragment may comprise a KMT2A:;AFFl-specific scTCR (e.g., single chain ⁇ TCR proteins such as V ⁇ -L-V ⁇ , V ⁇ -L-V ⁇ , V ⁇ -C ⁇ - L-V ⁇ , or V ⁇ -L-V ⁇ -C ⁇ , wherein V ⁇ and V ⁇ are TCR ⁇ and ⁇ variable domains respectively, C ⁇ and C ⁇ are TCR ⁇ and ⁇ constant domains, respectively, and L is a linker), which can be derived from the KMT2A::AFF1 TCR herein.
  • KMT2A:;AFFl-specific scTCR e.g., single chain ⁇ TCR proteins such as V ⁇ -L-V ⁇ , V ⁇ -L-V ⁇ , V ⁇ -C ⁇ - L-V ⁇ , or V ⁇ -L-V ⁇ -C ⁇ , wherein V ⁇ and V ⁇ are TCR ⁇ and ⁇ variable domains respectively, C ⁇ and C ⁇ are TCR ⁇
  • a “linker” may refer to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g., scTCR) to a target molecule or retains signaling activity (e.g., TCR complex).
  • a linker is composed of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids.
  • the linker may be composed of glycine residues, serine residues or a combination thereof.
  • the recombinant TCR of the invention is capable of binding or specifically binds to a KMT2A::AFF1 antigen peptide.
  • a "KMT2A::AFF1 antigen peptide” is a naturally or synthetically produced portion of a KMT2A::AFF1 antigen protein ranging in length from about 7 amino acids to about 15 amino acids, which can form a complex with a MHC (e.g., HLA) molecule and such a complex can bind with a TCR specific for a KMT2A::AFF1 antigen:MHC (e.g., HLA) complex.
  • MHC e.g., HLA
  • APC antigen presenting cells
  • MHC major histocompatibility complex
  • processed antigen peptides originating in the cytosol are generally from about 7 amino acids to about 11 amino acids in length and will associate with class I MHC molecules
  • peptides processed in the vesicular system e.g., bacterial, viral
  • KMT2A::AFF1 is an internal host protein
  • KMT2A::AFF1 antigen peptides will be presented in the context of class I MHC.
  • a KMT2A::AFF1 antigen peptide is RIRVDFKQTYSNEVH (SEQ ID NO:85), which is known to associate with human class I HLA, in particular with allele HLA- DPA1*02:01_DPBl*01:01.
  • the recombinant TCR of the invention is capable of binding or specifically binds to a KMT2A::AFF1 antigen peptide having the amino acid sequence of RIRVDFKQTYSNEVH (SEQ ID NO:85).
  • a recombinant TCR (TCR) specific for the KMT2A::AFF1 antigen as described herein includes variant polypeptide species that have one or more amino acid substitutions, insertions, or deletions in the amino acid sequence relative to the sequences of SEQ ID NOs:1-
  • TCR retains or substantially retains its specific binding function.
  • Conservative substitutions of amino acids are well known and may occur naturally or may be introduced when the TCR is recombinantly produced.
  • a "conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433; Lehninger (1975) Biochemistry, 2 nd Edition; Worth Publishers, Inc., NY, pp. 71-77; Lewin (1990) Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8).
  • Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired.
  • random or saturation mutagenesis techniques such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide- directed mutagenesis may be used to prepare immunogen polypeptide variants (see, e.g., Sambrook et al., supra).
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • a mutation is a substitution of one or three codons or amino acids, a deletion of one to about 5 codons or amino acids, or a combination thereof.
  • V ⁇ riants or mutants of a particular recombinant TCR specific for the KMT2A::AFF1 antigen may include a protein that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity, or any value or range therebetween) to any of the exemplary amino acid sequences disclosed herein (e.g., SEQ ID NOs:l-2 and/or SEQ ID NO:81-84), provided that (a) the CDRs have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof, and (b) the variant or mutant retains its ability to bind to the KMT2A::AFF1, e.g.
  • the present invention provides a TCR including an a-chain variable domain with a CDR3 having at least 90% sequence identity to an amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and/or a ⁇ -chain variable domain having at least 90% sequence identity to an amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2); wherein the TCR is capable of binding to the amino acid sequence RIRVDFKQTYSNEVH (SEQ ID NO:85) .
  • Sequence identity refers to the percentage of amino acid residues in one sequence that are identical with the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the percentage sequence identity values can be generated using the NCBI BLAST2.0 software as defined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, with the parameters set to default values.
  • the recombinant TCR of the invention, or the antigen binding fragment thereof is chimeric, humanized, or human.
  • a chimeric TCR refers to a chimeric antigen receptor (CAR).
  • CARs may be composed of three regions: an ectodomain, a transmembrane domain and an endodomain.
  • An ectodomain is the region of the receptor that is exposed to the extracellular fluid and is composed of the antigen binding domain described herein.
  • the transmembrane domain may be a hydrophobic domain that spans the membrane, e.g., a CD3-zeta transmembrane domain.
  • a transmembrane domain allows for incorporation of an artificial TCR into a native TCR complex.
  • a transmembrane domain includes a CD28 transmembrane domain.
  • An endodomain can be a functional intracellular portion of a receptor, such as a TCR or CAR.
  • an endodomain includes at least one, two or three ITAMs.
  • an endodomain includes a CD28 intracellular domain, an 0X40 intracellular domain, a CD3--zeta intracellular domain, or a chimeric intracellular domain thereof.
  • the invention provides a composition including a recombinant KMT2A::AFF1-specific TCR according to any one of the aforementioned aspects and a pharmaceutically acceptable carrier, diluent, or excipient.
  • Methods useful for isolating and purifying recombinantly produced TCR may include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant soluble TCR into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin.
  • One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated/recombinant soluble TCR described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR may be performed according to methods described herein and known in the art.
  • nucleic acids that encode the ⁇ -chain variable domain and/or ⁇ -chain variable domain of the TCR described herein.
  • polynucleotide “ “nucleic acid” or “nucleic acid molecule” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any of ligation, scission, endonuclease action, or exonuclease action.
  • the nucleic acids of the present disclosure are produced by PCR.
  • Nucleic acids may be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., ⁇ -enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
  • Nucleic acid molecules can be either single stranded or double stranded.
  • Isolated or recombinant polynucleotides or nucleic acid molecules encoding the TCR specific for KMT2A::AFF1 as described herein may be produced and prepared according to various methods and techniques known in the art.
  • a nucleic acid or polynucleotide may refer to a single- or a double-stranded DNA, cDNA or RNA in any form, and may include a positive and a negative strand of the nucleic acid which complement each other, including anti-sense DNA, cDNA and RNA. Also included are siRNA, microRNA, RNA-DNA hybrids, ribozymes, and other various naturally occurring or synthetic forms of DNA or RNA. [0037] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • the invention provides an isolated polynucleotide encoding a recombinant T cell receptor, or antigen binding fragment thereof, including an ⁇ -chain variable domain having a CDR3 amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and/or a ⁇ -chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2).
  • the polynucleotide further encodes an ⁇ -chain constant domain and/or a ⁇ -chain constant domain as described herein.
  • the ⁇ -chain variable domain and ⁇ -chain variable domain (and optional constant chains) of the TCR are encoded by separate nucleic acid molecules.
  • the a-chain variable domain and ⁇ -chain variable domain (and optional constant chains) of the TCR are encoded by the same nucleic acid molecule.
  • a nucleic acid molecule encoding both the a-chain variable domain and ⁇ -chain variable domain (and optional constant chains) of the TCR of the present invention further encodes a self-cleaving peptide disposed between the a-chain variable domain and ⁇ -chain variable domain.
  • the polynucleotides of the invention are provided in a vector.
  • a vector may include a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked, or which is capable of replication in a host organism.
  • vectors include plasmids, viral vectors, cosmids, and others.
  • Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell upon introduction into the host cell and thereby replicate along with the host genome. Additionally, some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as "expression vectors").
  • agents e.g., polynucleotides encoding recombinant TCRs specific for KMT2A: :AFF1, or variants thereof, as described herein
  • each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent) may be introduced to a cell or cell population or administered to a subject.
  • expression vector refers to a DNA construct containing a nucleic acid molecule that is operably- linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • a polynucleotide is codon optimized for efficient expression in a target host cell.
  • "plasmid,” “expression plasmid,” “virus” and “vector” are often used interchangeably.
  • expression refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.
  • the vector is a viral vector.
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and doublestranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox) .
  • ortho-myxovirus e.g., influenza virus
  • rhabdovirus e.g., rabies and vesicular s
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus , and hepatitis virus, for example.
  • retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin (1996) Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia).
  • the viral vector is a lentiviral vector or a retroviral vector.
  • Lentiviral vector means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.
  • the polynucleotide encoding the recombinant TCR specific for KMT2A::AFF1 may be operatively linked to certain elements of a vector.
  • polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are Ligated may be operatively linked.
  • operably- linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably-linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
  • Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control expression of the gene of interest.
  • Construction of one or more expression vectors that are used for recombinantly producing a recombinant TCR specific for the KMT2A::AFF1 may be accomplished by using any suitable molecular biology engineering techniques known in the art, including the use of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. ((2003) Current Protocols in Molecular Biology).
  • a polynucleotide in a recombinant expression construct includes at least one appropriate expression control sequence (also called a regulatory sequence), such as a leader sequence and particularly a promoter operably (i.e., operatively) linked to the polynucleotide sequence encoding the TCR.
  • a regulatory sequence also called a regulatory sequence
  • a promoter operably (i.e., operatively) linked to the polynucleotide sequence encoding the TCR.
  • the polynucleotide sequence encoding the TCR or recombinant expression vector comprising the same is delivered to an appropriate cell, for example, a T cell.
  • the recombinant expression vectors may also include, for example, lymphoid tissue-specific transcriptional regulatory elements (TRE) such as a B lymphocyte or T lymphocyte cell specific TRE. Lymphoid tissue specific TREs are known in the art (see, e.g., Thompson et al. (1992) Mol. Cell. Biol. 12:1043; Todd et al. (1993) J. Exp. Med. 177:1663; Penix et al. (1993) J. Exp. Med. 178:1483).
  • TRE lymphoid tissue-specific transcriptional regulatory elements
  • This invention also relates to host cells that express the recombinant KMT2A::AFF1-specific TCR on their cell surface.
  • Expression of the recombinant TCR in the host cell may be via a vector (e.g., expression vector) described herein or by introduction of the polynucleotides into the genome of the host cells (e.g., using CRISPR-Cas systems).
  • a vector e.g., expression vector
  • CRISPR-Cas systems CRISPR-Cas systems
  • Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. For example, see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory).
  • vectors and/or nucleic acid molecules encoding the KMT2A::AFF1-specific TCR are introduced into a host cell, e.g., a T cell used in adoptive immunotherapy.
  • the host cell used in such a therapy is allogeneic, syngeneic, or autologous.
  • Methods for introducing nucleic acids into T-cells have been described (e.g., US 2004/0087025), as have adoptive transfer procedures using T-cells of desired antigen-specificity (e.g., Schmitt et al. (2009) Hum. Gen. 20:1240; Dossett et al., Mol. Ther.
  • the term "introduced” in the context of inserting a nucleic acid molecule into a cell means “transfection", or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
  • transiently expressed e.g., transfected mRNA
  • the term "host cell” refers to a cell (e.g., T cell) or microorganism targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., KMT2A: :AFF1-specific TCR).
  • a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to biosynthesis of the heterologous or exogenous protein (e.g., inclusion of a detectable marker; deleted, altered or truncated endogenous TCR; increased co-stimulatory factor expression).
  • a heterologous or exogenous nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell.
  • the source of the heterologous or exogenous nucleic acid molecule, construct or sequence may be from a different genus or species.
  • a heterologous or exogenous nucleic acid molecule is added (i.e., not endogenous or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, or the like, wherein the added molecule may integrate into the host genome or exist as extra- chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may be present in multiple copies.
  • heterologous refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.
  • heterologous or exogenous nucleic acid molecule may be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
  • a host cell may be modified to express two or more heterologous or exogenous nucleic acid molecules encoding desired TCR specific for a KMT2A::AFF1 neoantigen (e.g., TCR ⁇ and TCR ⁇ ).
  • the two or more exogenous nucleic acid molecules may be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof.
  • the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.
  • the host cell expresses on its cell surface the recombinant KMT2A::AFF1-specific TCR, or antigen binding fragment thereof, which includes an a-chain variable domain having a CDR3 amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and/or a ⁇ -chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2).
  • the host cell has been genetically modified to have reduced cell surface expression of the endogenous TCR or no expression of an endogenous T cell receptor.
  • the term '''endogenous" or “native” refers to a gene, protein, or activity that is normally present in a host cell.
  • Gene-editing nucleases may be employed in order to disrupt components of the endogenous TCR. Since the TCR ⁇ / ⁇ dimer can produce a fully functioning TCR complex, disrupting TCR ⁇ and/or TCR ⁇ function may reduce (even eliminate) endogenous TCR expression. V ⁇ rious methods may be used to disrupt endogenous TCR ⁇ or TCR ⁇ genes. For example, four classes of gene editing proteins exist that share a common mode of action in binding a user defined sequence of DNA and mediating a double stranded DNA break (DSB). Zinc finger nucleases (ZFN) are heterodimeric arrays that co-localize at a target DNA site.
  • DSB double stranded DNA break
  • ZFNs include individual finger subunits that bind DNA and are tethered to the Fokl nuclease domain that cleaves DNA.
  • Transcription activator-like effector nucleases include repeating units that bind DNA by virtue of a hypervariable two amino acid sequence (repeat variable diresidue; RVD) that governs DNA base recognition. Similar to ZFNS, TALENs function as dimeric proteins that are fused to the Fokl endonuclease domain for DSB generation.
  • Meganucleases are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site.
  • the clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas9 nuclease platform involves a small guide RNA (gRNA) transcript that contacts a target DNA sequence via Watson-Crick base pairing and the Cas9 nuclease that cleaves the DNA.
  • gRNA small guide RNA
  • Any one or more of these gene editing enzymes may be used to disrupt expression of an endogenous TCR ⁇ and/or TCR ⁇ to disrupt assembly of the endogenous TCR ⁇ and TCR ⁇ .
  • a host cell is a human hematopoietic progenitor cell or human immune system cell transduced with a heterologous or exogenous nucleic acid molecule encoding a TCR ⁇ chain and/or a TCR ⁇ chain specific for a KMT2A::AFF1 antigen or antigen binding fragment thereof.
  • a "hematopoietic progenitor cell” is a cell that may be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cells types (e.g., immune system cells).
  • Exemplary hematopoietic progenitor cells include those with a CD24 Lo Lin CD117+ phenotype or those found in the thymus (referred to as progenitor thymocytes).
  • an "immune system cell” means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which gives rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which gives rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
  • a myeloid progenitor cell which gives rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes
  • lymphoid progenitor cell which gives rise to lymphoid
  • Exemplary immune system cells include a CD4 + T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a yd T cell, a regulatory T cell, and a natural killer cell.
  • Macrophages and dendritic cells may be referred to as "antigen presenting cells” or "APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.
  • the immune system cell is a T cell, a natural killer cell, or a combination thereof.
  • T cell is an immune system cell that matures in the thymus and produces TCRs.
  • T cells can be naive (i.e., not exposed to antigen and exhibit elevated expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO as compared to central memory T cells); memory T cells (T M ) (antigen-experienced and long-lived); and effector cells (antigen-experienced, cytotoxic).
  • T M may be further divided into subsets of central memory T cells (T CM , which exhibit increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cells) and effector memory T cells (T EM , which exhibit decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or T CM ).
  • Effector T cells (T E ) refer to antigen- experienced CD8 + cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for granzyrae and perforin as compared to T CM .
  • Other exemplary T cells include regulatory T cells, such as CD4 + CD25+ (Foxp3 + ) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8 + CD28”, and Qa-1 restricted T cells.
  • T cells may be obtained from various lymphoid tissues.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen tissue, lymphoid tissue, and tumors.
  • PBMCs peripheral blood mononuclear cells
  • the term "peripheral blood lymphocytes" (PBL) and its grammatical equivalents as used herein can refer to lymphocytes that circulate in the blood (e.gr., peripheral blood).
  • Peripheral blood lymphocytes can refer to lymphocytes that are not localized to organs.
  • Peripheral blood lymphocytes can include T cells, NK cells, B cell, or any combinations thereof.
  • T cells may be prepared according to methods known in the art.
  • T cells may be isolated from a subject.
  • T cells may be obtained from T cell lines.
  • T cells may be obtained from autologous sources.
  • T cells may be obtained from allogeneic sources.
  • T cells may also be obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
  • T cells may be an enriched T cell preparation, an APC-depleted cell preparation, or a substantially purified T cell preparation.
  • T cells may be a mixed T cell population or a purified T cell subset.
  • T cells may be an enriched T cell preparation containing a number or percentage of T cells that is increased with respect to an isolated population of T cells.
  • the T cells may be cultured under conditions effective for expanding the population of T cells.
  • T cell purification may be achieved, for example, by positive or negative selection including, but not limited to, the use of antibodies directed to CD2, CD3, CD4, CD5, CD8, CD14, CD19, and/or MHC class II molecules.
  • a specific T cell subset such as CD28 + , CD4 + , CD8+, CD45RA+, and/or CD45RO+ T cells, may be isolated by positive or negative selection techniques.
  • CD3 + , CD28 + T cells can be positively selected using CD3/CD28-conjugated magnetic beads.
  • enrichment of a T cell population by negative selection may be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • non-functional markers may be used to isolate T cells since binding of functional markers such as binding of CD3 by anti-CD3 antibody (alone or conjugated to magnetic particles) may trigger unwanted signaling events on T cells. Therefore, a cocktail of antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a (glycophorin A) may be used to isolate T cells.
  • a T cell sample may include cells from a subject's circulating blood and may be obtained by apheresis or leukopheresis.
  • a T cell sample may contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets.
  • Undesirable components of the T cell sample may be removed and the remaining T cells may be suspended in culture media.
  • cells may be washed to remove the plasma fraction.
  • T cells may be isolated from peripheral blood lymphocytes by lysing the red blood cells and by centrifugation through a PERCOLLTM gradient.
  • Nucleic acids encoding the recombinant TCR or vectors containing such nucleic acids may subsequently be delivered to the isolated T cells for expression and processing.
  • Recombinant T cells expressing the TCR may be cultured under conditions effective for expanding the population of T cells.
  • the invention also provides methods for treating a hyperproliferative disorder or a condition characterized by KMT2A::AFF1 neoantigen expression.
  • the invention provides an adoptive immunotherapy method for treating a subject diagnosed with, suspected of having or at risk for developing or recurrence of leukemia, wherein the leukemia expresses KMT2A::AFF1 antigen.
  • the terms, "treat” and "treatment,” refer to medical management of a disease, disorder, or condition of a subject (i.e., patient, host, who may be a human or non-human animal).
  • the method of the invention provides for administering to a subject in need of such treatment an appropriate dose and treatment regimen of a host cell expressing a recombinant KMT2A: :AFF1-specific TCR, or antigen binding fragment thereof, in an amount sufficient to provide therapeutic or prophylactic benefit.
  • Therapeutic or prophylactic benefit resulting from such treatment include, for example, an improved clinical outcome, wherein the object is to prevent or retard or otherwise reduce (e.g., decrease in a statistically significant manner relative to an untreated control) an undesired physiological change or disorder, or to prevent, retard or otherwise reduce the expansion or severity of such a disease or disorder.
  • Beneficial or desired clinical results from treating a subject include abatement, lessening, or alleviation of symptoms that result from or are associated the disease or disorder to be treated; decreased occurrence of symptoms; improved quality of life; longer disease-free status (i.e., decreasing the likelihood or the propensity that a subject will present symptoms on the basis of which a diagnosis of a disease is made); diminishment of extent of disease; stabilized (i.e., not worsening) state of disease; delay or slowing of disease progression; amelioration or palliation of the disease state; and remission (whether partial or total), whether detectable or undetectable; or overall survival.
  • Treatment can also mean prolonging survival when compared to expected survival if a subject were not receiving treatment .
  • Subjects in need of the methods and compositions described herein include those who already have the disease or disorder, as well as subjects prone to have or at risk of developing the disease or disorder.
  • Subjects in need of prophylactic treatment include subjects in whom the disease, condition, or disorder is to be prevented (i.e., decreasing the likelihood of occurrence or recurrence of the disease or disorder) .
  • the presence or risk of developing or recurrence of a leukemia in a subject may be diagnosed and classified by established methods.
  • the clinical benefit provided by the compositions (and preparations comprising the compositions) and methods described herein can be evaluated by design and execution of in vitro assays, preclinical studies, and clinical studies in subjects to whom administration of the compositions is intended to benefit.
  • the leukemia being treated in accordance with the adoptive immunotherapy method of this invention includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), or mixed phenotypic leukemia, and subtypes thereof.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • mixed phenotypic leukemia and subtypes thereof.
  • Subjects being treatment may include, for example, mammals, humans, pregnant women, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, newborn, or neonates.
  • a subject may be a patient.
  • a subject may be a human.
  • a subject may be a child (i.e., a young human being below the age of puberty).
  • a subject may be an infant.
  • the subject may be a formula-fed infant.
  • a subject may be an individual enrolled in a clinical study.
  • a subject may be a laboratory animal, for example, a mammal, or a rodent.
  • the subject may be a mouse.
  • the subject has previously been treated with one or more different cancer treatment modalities. In some embodiments, the subject has previously been treated with one or more of radiotherapy, chemotherapy, or immunotherapy. In some aspects, the subject has been treated with one, two, three, four, or five lines of prior therapy .
  • Host cells expressing the recombinant TCR specific for the KMT2A::AFF1 antigen as described herein may be administered to a subject in a pharmaceutically or physiologically acceptable or suitable excipient or carrier.
  • Pharmaceutically acceptable excipients are biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject.
  • a therapeutically effective dose is an amount of host cells (expressing a recombinant TCR specific for KMT2A::AFF1 antigen) used in adoptive transfer that is capable of producing a clinically desirable result (i.e., a sufficient amount to induce or enhance a specific T cell immune response against cells expressing KMT2A: :AFF1 antigen (e.g., a cytotoxic T cell response) in a statistically significant manner) in a treated human or non-human mammal.
  • a clinically desirable result i.e., a sufficient amount to induce or enhance a specific T cell immune response against cells expressing KMT2A: :AFF1 antigen (e.g., a cytotoxic T cell response) in a statistically significant manner
  • the dosage for any one patient depends upon many factors, including the patient's size, weight, body surface area, age, the particular therapy to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a preferred dose for administration of a host cell comprising a recombinant expression vector as described herein is about 10 7 cells/m 2 , about 5*10 7 cells/m 2 , about 10 8 cells/m 2 , about 5x10 8 cells/m 2 , about 10 9 cells/m 2 , about 5 ⁇ 10 9 cells/m 2 , about 10 10 cells/m 2 , about 5 ⁇ 10 10 cells/m 2 , or about 10 11 cells/m 2 .
  • compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art.
  • An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration.
  • an appropriate dose and treatment regimen provide the composition (s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity).
  • a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.
  • Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre- clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
  • Certain methods of treatment or prevention contemplated herein include administering a host cell (which may be autologous, allogeneic or syngeneic) comprising a desired nucleic acid molecule as described herein that is stably integrated into the chromosome of the cell.
  • a host cell which may be autologous, allogeneic or syngeneic
  • a desired nucleic acid molecule as described herein that is stably integrated into the chromosome of the cell.
  • a cellular composition may be generated ex vivo using autologous, allogeneic or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) in order to administer a desired, KMT2A::AFF1 antigen- targeted T-cell composition to a subject as an adoptive immunotherapy.
  • the host cells are expanded before and/or after introduction of the desired nucleic acid molecule .
  • administration of a composition or therapy refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state.
  • the host cell is co-administered with an adjunctive therapy (e.g., a cytokine such as IFN- ⁇ , IL-2, IL-15, IL-21, or any combination thereof).
  • an adjunctive therapy e.g., a cytokine such as IFN- ⁇ , IL-2, IL-15, IL-21, or any combination thereof.
  • Such coadministration may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., KMT2A::AFF1 antigen-specific recombinant host cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof).
  • the subject being treated has received a non-myeloablative or a myeloablative hematopoietic cell transplant, wherein the treatment may be administered at least two to at least three months after the non-myeloablative hematopoietic cell transplant.
  • An effective amount of a therapeutic or pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein.
  • An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount" may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.
  • a disease or disease-state e.g., recurrence
  • the level of a cytotoxic T lymphocyte (CTL) immune response may be determined by any one of numerous immunological methods described herein and routinely practiced in the art.
  • the level of a CTL immune response may be determined prior to and following administration of the KMT2A::AFF1 antigen-specific TCR expressed by, for example, a T cell.
  • Cytotoxicity assays for determining CTL activity may be performed using any one of several techniques and methods routinely practiced in the art (see, e.g., Henkart et al. (2003) "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) Lippincott Williams & Wilkins, Philadelphia, PA, pages 1127-50).
  • Antigen-specific T cell responses are typically determined by comparisons of observed T cell responses according to any of the herein described T cell functional parameters (e.g., proliferation, cytokine release, CTL activity, altered cell surface marker phenotype, etc.) that may be made between T cells that are exposed to a cognate antigen in an appropriate context (e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells) and T cells from the same source population that are exposed instead to a structurally distinct or irrelevant control antigen.
  • a cognate antigen e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells
  • a response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.
  • a biological sample may be obtained from a subject for determining the presence and level of an immune response to a KMT2A::AFF1 antigen-specific TCR as described herein.
  • a "biological sample” as used herein may be a blood sample (from which serum or plasma may be prepared), biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source.
  • Biological samples may also be obtained from the subject prior to receiving any composition, which biological sample is useful as a control for establishing baseline (i.e., pre-adoptive immunotherapy) data.
  • compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until.
  • a unit dose includes a recombinant host cell as described herein at a dose of about 10 7 cells/m 2 to about 10 11 cells/m 2 .
  • the composition may also include sterile aqueous or oleaginous solution or suspension.
  • suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic salt solution, 1,3-butanediol, ethanol, propylene glycol or polyethylene glycols in mixtures with water.
  • Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate.
  • any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired therapeutic effect in association with an appropriate pharmaceutical carrier.
  • an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects.
  • Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine in the art and may be performed using samples obtained from a subject before and after treatment.
  • Leukemia Specimens The leukemia specimens were used in accordance with the ethical approval granted by the Institutional Review Board of St. Jude Children's Research Hospital. Informed consent was obtained from the patients, parents, or guardians, as appropriate. Bone marrow or peripheral blood was collected at diagnosis and/or relapse. Mononuclear cells were isolated by density-gradient centrifugation and cryopreserved until analysis. The patient characteristics are provided in Tables 1-2.
  • T cells were expanded based on the REP method (Gros et al. (2016) Nat. Med. 22(4):433- 438). In brief, T cells were maintained in culture with irradiated (5,000 rad) allogeneic PBMC feeder cells from 3 healthy donors. The T-cell media were supplemented with 30 ng/mL anti-CD3 OKT3 and 3,000 10/mL IL2. After 6 days, the culture medium was replenished with fresh T-cell media containing IL2 every 2 days. At Day 15, the expanded T cells were either used in co-culture assays or cryopreserved.
  • T-Cell Reactivity IFN-y ELISPOT Assay and Detection of Activation Markers by Flow Cytometry. T-cell reactivity was assessed by IFN- ⁇ secretion and upregulation of 4-1BB and OX40 on CD8+ and CD4 + T cells, respectively (Gros et al. (2016) Nat. Med. 22 (4):433-438; Parkhurst et al. (2017) Clinical Cancer Research 23(10):2491-2505; Tran et al. (2015) Science 350 (6266):1387-1390). IFN- ⁇ secretion was measured by ELISPOT.
  • the cells were collected and stained for 4-1BB and 0X40 for flow cytometry analysis, as previously described (Gros et al. (2016) Nat. Med. 22(4);433-438; Tran et al. (2015) Science 350 (6266):1387-1390).
  • the assessments were performed by co- culture of 100,000 target cells and T cells for 20 hours. The number of T cells differed based on their sources: 100,000 cells were used when expanded T cells from primary leukemia material were used, and 20,000 cells were used when specific candidate TCR T cells were used. Target cells and T cells were washed to remove excess cytokines before co-culture. The co- cultures were performed in T-cell media without exogenous cytokines.
  • Plate-bound anti-CD3 OKT3 was used as the positive control. Media without T cells, irrelevant TCRs, and irrelevant peptides were used as negative controls. Positivity was considered when the readout values were greater than a 2- fold increase over that of the background and more than 40 spots were detected in the ELISPOT assay. The experiments were performed in duplicate, unless otherwise specified, due to the limited availability of the primary leukemia specimens.
  • PDX Specimens Leukemic PDX samples (SJAML001441 and SJAML030459) were obtained through the St. Jude Children's Research Hospital Public Resource of Patient-derived and Expanded Leukemias (PROPEL). Briefly, PDX were established by tail vein injection of primary leukemia cells into eight- twelve-week-old sub-lethally irradiated (250 Rad) NOD.Cg- Prkdc scid ⁇ I12rg tm1Wjl Tg(CMV-7 IL3,CSF2,KITLG)1Eav/MloySzJ (NSG- SGM3) (The Jackson Laboratory).
  • Spleen cells harvested from engrafted mice were used for expansion in subsequent passages. The level of engraftment was monitored by monthly retro- orbital bleeds and flow cytometric analysis of human CD45 positive cells. The study was approved by the St. Jude Children's Research Hospital Animal Care and Use Committee and carried out according to Office of Laboratory Animal Welfare guidelines .
  • Culture media were supplemented with 20 ng/mL interleukin 3 (IL3) and 10 ng/mL IL7 for ALL samples, or with SCF, TPO, IL3, IL6, and FLT3L (10 ng/mL each) for AML and MPAL samples. After 7 days in culture, samples were transferred to a new layer of feeder cells.
  • IL3 interleukin 3
  • APCs B cells were used as APCs and were generated from PBMCs obtained during the patient's remission state. The cells were isolated using CD19 + microbeads (Miltenyi Biotec) and maintained in culture with irradiated NIH3T3 CD40L feeder cells, as previously described (Tran et al. (2015) Science 350(6266):1387-1390; Gros et al. (2016) Nat. Med. 22(4):433-438) .
  • IMDM Iscove's Modified Dulbecco's Medium
  • human serum AB 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 2 mM L-glutamine, and 200 U/mL IL4 (Peprotech).
  • the cells were either used in co-culture assay or cryopreserved.
  • cryopreserved samples were used, the B cells were thawed and rested in B-cell media for 16-24 hours before the coculture assay (Gros et al. (2016) Nat. Med. 22 (4):433-438).
  • Antigen-reactive T lymphocytes were sorted based on CD8 + 4-lBB + and CD4 + 4-lBB + or CD4 + OX40 + , as previously described (Parkhurst et al. (2017) Clinical Cancer Research 23(10):2491-2505; Yossef et al. (2016) JCI Insight 3(19):el22467). Single cells were sorted into a 384-well plate and subsequently processed for targeted TRA and TRB sequencing. CD39 + and PD1 + T cells were sorted from primary leukemia blasts. PD1 + T cells were defined as the top 20% of the CD3 + population. The enriched cells were expanded using the REP culture system.
  • Antibodies used for T-cell reactivity assessment, cell sorting, and PD1 + population characterization included CD3 APC/H7 (RRID: AB 1645475), CD4 ALEXA FLUOR® 700 (RRID; AB,2563150), CD4 PE (RRID: AB_395752), CD8 PE/Cy7 (RRID: AB_396852), 0X40 FITC (RRID: AB___396160), 4-1BB APC (RRID: AB 398477), CD39 BV421 (RRID: 48 AB_2564575), PD1 PE (RRID: AB_940483), TIM3 BV650 (RRID: AB_2565829), and mTCRB PE (AB_466066).
  • Antibodies used for MHC expression analysis included CD19 PE/Cy5 (RRID: AB_314240), CD33 ALEXA FLUOR® 647 (RRID: AB_2927892), CD33 PE (RRID: AB_2566106), HLA-A,B,C PE/Cy5 (RRID: AB_314877), HLA-DR,DP,DQ APC/Fire 750 (RRID: AB 2750314), HLA-A BV711 (RRID: AB_2917819), HLA-B PE (RRID: AB_2916506), HLA-C ALEXA FLUOR® 647 (RRID: AB 2894582), and HLA-DR (RRID: AB__2561913).
  • T cells from healthy donor apheresis were activated using anti-CD3/CD28 DYNABEADS® (ThermoFisher) in RPMI supplemented with 10% FBS, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 2 mM L-glutamine, 10 mM HEPES, 50 lU/mL IL2, 10 ng/mL IL7, and 10 ng/mL IL15.
  • the cells were seeded at a concentration of 2 ⁇ 10 6 cells/mL and activated for 48 hours.
  • TCR Retrovirus Production and Transduction TRB and TRA sequences were joined by a furin SGSG P2A linker. Mouse constant regions were used (Haga-Friedman et al. (2012) J. Immunol. 188(11):5538-5546; Cohen et al. (2006) Cancer Res. 66 (17):8878-8886; Cohen et al. (2007) Cancer Res. 67(8):3898- 3903) .
  • the sequences were synthesized (Twist Bioscience) and cloned into a pSFG retroviral backbone.
  • the co-transfection was composed of 9 ⁇ g expression plasmid, 9 ⁇ g Peq-Pam, 4 ⁇ g RD114, and 60 ⁇ L LIPOFECTAMINE® 2000 transfection reagent.
  • the mixture was added to 293T cells in a 10-cm dish format or down-scaled in a 35-mm dish with the same ratio. Supernatant was collected at 24 and 48 hours, filtered using 0.45-pm pore size, and used for transduction or stored at -80°C. Transduced cells were used at Day 7.
  • B cells were washed and resuspended at a concentration of 5-10 x 10 6 cells in 100 ⁇ L optiMEMTM. Then 8 ⁇ g RNA was added to the cells, which were then transferred to a 2-mm gap cuvette and electroporated at 150 V, 20 ms, 1 pulse (Gros et al. (2019) J. Clin. Invest. 129(11):4992-5004) using a BioRad Gene Pulser. The B cells were incubated at 37°C, under 5% CO2, for 16 hours and used for co-culture assays.
  • Cytotoxicity Assay Cell-mediated killing assay was performed based on CFSE (carboxyfluorescein succinimidyl ester) and live/dead staining (Gros et al. (2019) J. Clin. Invest. 129(11):4992-5004).
  • Target cells (3 ⁇ 10 4 cells) were stained with 0.25 pM CFSE by incubating at 37°C, under 5% CO 2 , for 20 minutes. Cells were washed, then co-cultured with T cells at an effector-to-target ratio of 1:1, 2;1, or 8:1. The co-culture was conducted for 18 hours in a 96-well plate.
  • the cells were stained with Live/Dead Ghost DyeTM Violet 510 (Tonbo) for 30 minutes at room temperature. The cells were then washed and resuspended in staining buffer containing 5 ⁇ L CountBrightTM Absolute Counting Beads (ThermoFisher) per well. The samples were analyzed by flow cytometry, and cytotoxicity was calculated from duplicates. Target cells without T cells were used as the negative control, and 50 ⁇ g/mL blasticidin was used as the positive control.
  • Generating Lentiviral-Transduced K562 MHC class II APCs The MHC class II machinery cDNA sequences were synthesized by Genscript. The components were generated as 2 separate transgenes, including CD64, CD80, CD83, CD74, and HLA-DM. They were cloned into a pLVX lentiviral backbone and transduced into K562 cells. The cells were stained with fluorescent antibodies, and the population that expressed all components was isolated.
  • TCR ⁇ and TCR ⁇ chains were amplified using a 5' Rapid Amplification of cDNA Ends (RACE) with unique molecular identifiers (UMIs) for error correction, essentially as described (Egorov et al. (2015) J. Immunol. 194(12):6155-6163).
  • RACE 5' Rapid Amplification of cDNA Ends
  • UMIs unique molecular identifiers
  • RNA was extracted and reverse- transcribed using SmartScribeTM RT reagent (Takara), and Q5 polymerase (New England Biolabs) was used during first- and second-round amplifications.
  • TCR ⁇ and TCR ⁇ amplicons generated by the second-round PCR were pooled by equal volume, prepped, and indexed for sequencing on Illumina platforms using a KAPA HyperPrep Kit (Roche).
  • the 150-bp paired-end sequencing was performed on an Illumina NovaSeq6000 by the St. Jude Hartwell Center.
  • Blood or bone marrow samples were obtained from 34 patients at the time of diagnosis or relapse.
  • the cohort included 15 patients with ALL, 15 patients with AML, and 4 patients with MPAL.
  • Eight patients with ALL were infants with the KMT2A::AFF1 fusion.
  • Samples were curated based on whether their genetic mutations were predictors of poor prognosis (i.e., KMT2A-rearrangement, NUP98::NSD1, PICALM::MLLT10, or DEKt :NUP214), were highly prevalent ⁇ RUNX1::RUNX1T1), or were enriched in MPAL (e.g. ZNF384::EP300 in B/myeloid MPAL; Alexander et al.
  • T-cell expansion ranged from 133- to 4092-fold increase (median, 1027-fold) in ALL samples, 172- to 4957-fold increase (median, 907-fold) in AML, and 286- to 2693-fold increase (median, 554-fold) in MPAL.
  • T-cell killing by the expanded T cells was assessed. Specifically, killing of the autologous blasts in an infant (less than 1 years of age) ALL sample with KMT2A;;AFF1 (SJINF002), a pediatric ALL sample with KMT2A::AFF1 (SJALL016500; patient derived xenograft - PDX), and a pediatric ALL sample with PICALM: :MLLTlO (SJALL048457) was determined. All 3 samples demonstrated effective T-cell mediated killing of autologous leukemic blasts (FIG. 1), indicating T-cell reactivity in all 15 patients with ALL.
  • T-cell responses in 15 AML and 4 MPAL samples were subsequently evaluated. These samples included KMT2A- rearrangements, NUP98::NSD1, PICALM::MLLT10, RUNX1:RUNX1T1, and ZNF384::MLLT10 molecular alterations (Table 2).
  • the MPAL cases included 2 patient samples with myeloid/T-cell phenotypes (SJMPAL016107 and SJMPAL011911) and 2 patients with myeloid/B-cell phenotypes (SJMPAL012424 and SJMPAL012425).
  • the REP culture system was used to expand the T cells and then the expanded T cells were co-cultured with autologous leukemia blasts.
  • MHC class I and II expression after IFN- ⁇ pre-treatment was then evaluated. Both MHC class I and II were upregulated at a median of 1.4-fold (range, 0.9 to 15.7-fold) and 2.4-fold (range, 0.7 to 70.5-fold), respectively.
  • the expanded T cells were co-cultured with their autologous IFN- ⁇ treated AML or MPAL blasts; 4-1BB was upregulated on CD8 + and CD4 + populations in 13 AML cases, which correlated with an increase in IFN- ⁇ production and 0X40 expression by CD4 + T cells.
  • the AML sample SJMLL012 showed T-cell reactivity by IFN- ⁇ secretion and 0X40 on CD4 + T cells, despite no upregulation of the 4-1BB marker.
  • SJAML061496 showed no reactivity.
  • All 4 MPAL samples showed upregulation of 4-1BB and 0X40 and increased IFN- ⁇ secretion.
  • T-cell killing of the autologous blasts in 5 AML samples and 2 MPAL samples was also assessed.
  • Three of the 5 AML samples and both MPAL samples showed cytotoxicity (FIG. 2).
  • Both AML samples with the KMT2A::MLLT10 fusion gene showed no cytotoxicity (SJAML005142 and SJMLL012).
  • Example 5 Expression of PD1 or CD39 Marks Leukemia Blasts for T-Cell Recognition
  • PD1 ⁇ CD39+ and PD hi populations had up to 2.5-fold greater response to leukemia blasts, as shown by 4- IBB upregulation and increased IFN- ⁇ secretion. Furthermore, the PDl hi population co-expressed CD39 and TIM3 at higher levels than did the PDl dim or PD1- populations. CD39 and TIM3 were more highly expressed on T cells from the leukemia cohort, when compared to cells from healthy donors). These data indicate that the leukemia-reactive T-cell population can be isolated using these markers. Additionally, TIM3 is recognized as an AML stem cell marker, and expressed at higher levels in AML cells and ALL cells during disease progression. Together, these data indicate that TIM3 inhibitors can improve T-cell function and target leukemia blasts.
  • TCRs T cell receptors
  • the expanded T cells were co-cultured with autologous blasts, and then the leukemia-reactive T cells were enriched by sorting for 4-lBB + on the CD8 + population and for 4-1BB + , OX40 + , or 4-lBB + OX40 + on the CD4 + population.
  • Cells were sorted into a 384-well plate for single-cell TCR sequencing.
  • Infant ALL samples with a KMT2A::AFF1 fusion at diagnosis (SJINF013), or at relapse (SJINF002) and AML samples with PICALM::MLLT10 (SJAML030459), NUP98::NSD1 (SJAML001441), or RUNX1::RUNX1T1 (SJAML030471) fusions were included in this analysis.
  • infant ALL with W T2A-rearrangements has a mean of 1.3 non-silent mutations, which lowers the complexity of the neoantigen screening.
  • the top 4-6 clonotypes in these samples were selected to evaluate their reactivity to leukemia blasts and their fusion genes. Additionally, the top 10 clonotypes of SJAML030471 were selected, which did not show any major reactive expansions.
  • Healthy donor peripheral blood lymphocytes were used as an alternative to autologous peripheral blood mononuclear cells (PBMCs). Endogenous TCRs on healthy donor peripheral blood lymphocytes were knocked out by using CRISPR of the TRAC locus (Roth et al. (2016) Nature 559 (7714):405-409).
  • Candidate TCRs were then overexpressed by retroviral transduction. Fusion breakpoint sequences were obtained by PCR analysis of cDNA, followed by Sanger sequencing.
  • TCR SJINF002 clone 2 (ranked 2 by frequency), SJINF013 clone 2, SJAML001441 clone 2, and SJAML030459 clones 3 and 6 were identified as reacting to leukemia blasts and/or fusion neoantigen (FIGS. 3A-3E).
  • fusion neoantigen atypical fusion neoantigen
  • TCRs Antigen recognition and reactivity of these TCRs was further confirmed by performing cytotoxicity assays. The reactivity against primary leukemia SJINF002 and SJAML001441, and PDX SJAML030459 (due to limited sample availability) was tested. Peripheral blood lymphocytes transduced with reactive TCRs from samples SJINF002, SJAML001441, and SJAML030459 were co-cultured with leukemia blasts, and elimination of blasts was assessed. T cell-mediated killing of leukemic blasts in SJIN002 and SJAML001441 was observed, but not in SJAML030459 (FIG. 4).
  • TCR clonotype SJAML001441 was cytotoxic against leukemic blasts, despite not being reactive toward the fusion neoantigen, indicating recognition of another tumor-specific or associated antigen.
  • HLA human leukocyte antigen
  • HLA-restriction was also determined for reactive TCR clones 3 and 6 in sample SJAML030459. Index fluorescence- activated cell sorting (FACS) indicated that these clones originated from CD4 + and CD8 + T cells, respectively. Each HLA allele and the PICALM:tMLLTlO fusion were transfected into 293T-CIITA. The reactivity of TCR clones 3 and 6 was observed when this fusion neoantigen was presented on DRA*01 :01/DRB3*01:01 and HLA B*51:01, respectively, as indicated by 4-IBB or 0X40 upregulation and increased IFN- ⁇ secretion (FIGS. 5B-5C).
  • FACS Index fluorescence- activated cell sorting
  • Activation marker upregulation and increased IFN- y secretion of T cells expressing TCR SJINF002 clone 2 to the fusion peptide RIRVDFKQTYSNEVH indicated recognition of the fusion (FIG. 6).
  • the TCR SJINF002 clone 2 was present only at relapse (Day 1006), but at a low frequency (0.01%). Notably, this clonotype was enriched to 11.7% in the 4-1BB + OX40 + population.
  • Example 7 TCR SJINF002_2-expressing T Cells Reduce Tumor Burden and Improve survival
  • mice were transplanted with 1 million leukemia blasts SJINF002, and engraftment was monitored by weekly blood draws analyzed through fluorescence-activated cell sorting (FACS). Once engraftment was confirmed (at 0.1% blast levels), TCR SJINF0022 and an irrelevant control TCR were generated by retroviral transduction into TRAC knockout PBMCs from healthy donors. Five million TCR SJINF002_2-expressing T cells were subsequently infused intravenously. [0118] Each week, tumor burden and the persistence of the transferred T cells in the peripheral blood were monitored, as was as body weight, which served as a morbidity indicator. Starting from week 3 post-T cell transfer, a marked reduction in tumor burden (FIG.

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Abstract

Est divulgué un récepteur de lymphocytes T recombinant qui reconnaît le néo-antigène de fusion KMT2A::AFF1 présenté par HLA-DPAl*02 : 01 DPBl*01 : 01, des polynucléotides codant pour le récepteur de lymphocytes T recombinant, et un vecteur d'expression et une cellule hôte recombinante comprenant de tels polynucléotides. L'invention concerne également un procédé d'utilisation du récepteur de lymphocytes T recombinant en immunothérapie adoptive pour traiter la leucémie.
PCT/US2024/058390 2023-12-08 2024-12-04 Récepteur de lymphocytes t spécifique du néoantigène kmt2a::aff1 et son utilisation en immunothérapie adoptive Pending WO2025122569A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9803246B2 (en) * 2011-06-28 2017-10-31 International Institute Of Cancer Immunology, Inc. Receptor gene for peptide cancer antigen-specific T cell
WO2023232785A1 (fr) * 2022-05-30 2023-12-07 Hs Diagnomics Gmbh Récepteurs de lymphocytes t spécifiques à une tumeur communs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9803246B2 (en) * 2011-06-28 2017-10-31 International Institute Of Cancer Immunology, Inc. Receptor gene for peptide cancer antigen-specific T cell
WO2023232785A1 (fr) * 2022-05-30 2023-12-07 Hs Diagnomics Gmbh Récepteurs de lymphocytes t spécifiques à une tumeur communs

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