T cell receptor specific to KMT2a::AFF1 neoantigen and use thereof in adoptive immunotherapy

Abstract

Disclosed is a recombinant T cell receptor that recognizes the KMT2A: :AFF1 fusion neoantigen presented by HLA- DPAl*02 : 01 DPBl*01 : 01, polynucleotides encoding the recombinant T cell receptor, and an expression vector and recombinant host cell comprising such polynucleotides. A method of using the recombinant T cell receptor in adoptive immunotherapy for treating leukemia is also provided.

Description

T CELL RECEPTOR SPECIFIC TO KMT2A::AFF1 NEOANTIGEN AND USETHEREOF IN ADOPTIVE IMMUNOTHERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit from U.S. Provisional Patent Application Serial Nos. 63 / 607,642, filed December 8, 2023, the contents of which are incorporated herein by reference in their entireties.FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under grant nos. CA265009 and AI136514 awarded by the National Institutes of Health. The government has certain rights in this invention.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0003] A Sequence Listing in XML format, entitled SJ0114WO_ST26.xml, 75,572 bytes in size, generated on November 8, 2024, is filed herewith. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures.BACKGROUND OF THE INVENTION

[0004] Fusion-driven leukemias can be clinically aggressive and associated with poor prognosis. Despite the overall improvement in outcomes for pediatric patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and mixed phenotypic leukemia (MPAL) over the last several decades, some subtypes characterized by specific genetic fusions continue to be associated with poor outcome. For example, leukemias harboring certain KMT2A rearrangements canhave a dismal prognosis, with a 5-year event free survival less than 30%. To improve outcomes, agents that target fusion- associated pathways are currently being tested, including Menin and NSD1 inhibitors. Furthermore, 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.

[0005] 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. (2018) 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. These approaches are being investigated as therapeutic strategies after disease progression or as relapse prophylaxis during remission after hematopoietic stem cell transplantation. Moreover, the combination of chemotherapy and immune-checkpoint blockade has been introduced into AML treatment, with promising results. Furthermore, the hypomethylating agent, azacitidine, enhances immune responses by increasing interferon gamma (IFN-γ) production and major histocompatibility complex (MHC) expression and is used in combination with PD1 blockade in AML treatment with modest improvement in survival. Additional trials of combinations of nivolumab and pembrolizumab with conventional chemotherapy indicate that adding PDl-blockade also improves outcome. Altogether, the success of immunotherapies and other immune modulating agents in the treatment of leukemia correlates withimproved adaptive immune responses against leukemic blasts, particularly by T-cells.

[0006] Endogenous T-cell recognition of the ETV6::RUNX1 fusion neoantigen has been observed in patients with ALL and is the most common genetic aberration in childhood ALL, occurring in approximately 25% of patients (Zamora et al. (2019) Sci. Transl. Med. 11(498):eaat8549; Bhojwani et al. (2012) Leukemia 26(2):265-270). Furthermore, other studies have identified T-cell responses in leukemias that are characterized by BCR::ABL1 (Comoli et al. (2017) Blood 129 (5):582-586) or CBFB::MYH11 (Biernacki et al. (2020) J. Clin. Invest. 130(10):5127-5141) fusions. These fusions and others that characterize different leukemic subtypes occur early in leukemogenesis, even in pre-leukemic clones, and are essential for cell survival. For example, 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. (2009) Blood 113(20):4922-4929), RUNX1 ::RUNX1T1 (Martinez Soria et al. (2009) Leukemia 23 (1):188-190), and PML::RARA (Huang et al. (1988) Blood 72 (2):567-572), showed potent anti-leukemic activity. Together, these data indicate that targeting fusion genes has the potential to induce durable remissions.

[0007] Adoptive T-cell therapy using T-cells in the treatment of leukemia characterized by fusion oncoproteins are needed. The present invention meets this need in the art.SUMMARY OF THE INVENTION

[0008] 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 β-chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID N0:2). 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.

[0009] 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 providedBRIEF DESCRIPTION OF DRAWINGS

[0010] 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.

[0011] 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.

[0012] 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. 3B), TCR SJAML00144_2 (FIG. 3C), TCR SJAML030459_3 (FIG. 3D), and SJAML030459_6 (FIG. 3E). The reactivity was measured by IFN-γ 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).

[0013] 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.

[0014] 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.

[0015] 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-γ secretion (right panel) .

[0016] 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.DETAILED DESCRIPTION OF THE INVENTION

[0017] 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.

[0018] Accordingly, 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. Non-limiting examples of these and related uses are described herein and include in vitro, ex vivo and in vivo stimulation of KMT2A::AFF1 neoantigen-specific T-cell responses, such as by the use of recombinant T cells expressing a TCR specific for a KMT2A::AFF1 neoantigen.

[0019] As used herein, "T cell receptor" (TCR) 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. (1997) Immunobiology: The Immune System in Health and Disease, 3rdEd., Current Biology Publications, p. 4:33) capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is composed of a heterodimer having α and β chains (also known as TCRα and TCRβ, respectively), or γ and δ chains (also known as TCRγ and TCRδ, respectively). Like immunoglobulins, the extracellular portion of TCR chains (e.g., α-chain, β-chain) contain two immunoglobulin domains, a variable domain (e.g., α-chain variable domain or Vα, β- 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., α-chain constant domain or Cα, typically amino acids 117 to 259 based on Kabat, β-chain constant domain or Cβ, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. Also like immunoglobulins, the 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). In some aspects, 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.

[0020] In some aspects, the recombinant TCR is from a human. In certain aspects, 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 orrange 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] In some aspects, the recombinant TCR of the invention includes a β-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 β-chain constant domain having an amino acid sequence as set forth in SEQ ID NO:83 or SEQ ID NO:84 :

[0022] In some aspects, the invention provides a recombinant TCR, or antigen binding fragment thereof, that specifically binds to or is specific for a KMT2A::AFF1 fusion neoantigen. As used herein, a "KMT2A: :AFF1 fusion neoantigen" or "KMT2A: :AFF1 antigen" refers to the neoantigen resulting from the translocation t(4;11) (q21;q23). It accounts forapproximately 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. The wild-type gene sequences encoding 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. In some aspects, the KMT2A: :AFF1 antigen results from a breakpoint located at exons 10 and 4, respectively.

[0023] As used herein, "specifically binds" or "specific for" refers to an association or union of a binding protein (e.g., TCR) to a target molecule, while not significantly associating or uniting with any other molecules or components in a sample. 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 Kaof at least about 107M-1, at least about 108M"1, at least about 109M-1, at least about 1010M-1, at least about 1011M-1, at least about 1012M-1or at least about 1013M-1. "Low affinity" binding proteins refer to those binding proteins or binding domains having a Kaof up to about 107M-1, up to about 106M-1, up to about 105M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., about 10-5M to about 10-13M).

[0024] 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)Cancer Res. 53:2560; and US 5,283,173 or US 5,468,614).

[0025] In some aspects, 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%). In other aspects, the invention provides a recombinant KMT2A::AFF1-specific TCR, or antigen binding fragment thereof, including a β-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%). In further aspects, 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 β-chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2). In further aspects, 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 β-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 β-chain constant domain having an amino acid sequence of SEQ ID NO:83 or SEQ ID NO:84.

[0026] 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::AFFl-specificantigen 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 10-8M, less than about 10-9M, less than about 10-10M, less than about 10-11M, less than about 10-12M, or less than about 10-13M.

[0027] In some aspects, an KMT2A::AFF1 antigen binding fragment may comprise a KMT2A:;AFFl-specific scTCR (e.g., single chain αβTCR proteins such as Vα-L-Vβ, Vβ-L-Vα, Vα-Cα- L-Vα, or Vα-L-Vβ-Cβ, wherein Vα and Vβ are TCRα and β variable domains respectively, Cα and Cβ are TCRα and β constant domains, respectively, and L is a linker), which can be derived from the KMT2A::AFF1 TCR herein. For the purposes of this invention, 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). In certain aspects, 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. In some aspects, the linker may be composed of glycine residues, serine residues or a combination thereof.

[0028] In some aspects, 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 TCRspecific for a KMT2A::AFF1 antigen:MHC (e.g., HLA) complex. Principles of antigen processing by antigen presenting cells (APC) (such as dendritic cells, macrophages, lymphocytes or other cell types), and of antigen presentation by APC to T cells, including major histocompatibility complex (MHC)- restricted presentation between immunocompatible (e.g., sharing at least one allelic form of an MHC gene that is relevant for antigen presentation) APC and T cells, are well established (see, e.g., Murphy, Janeway's Immunobiology (8thEd.) 2011 Garland Science, NY; chapters 6, 9 and 16). For example, processed antigen peptides originating in the cytosol (e.g., tumor antigen, intracellular pathogen) are generally from about 7 amino acids to about 11 amino acids in length and will associate with class I MHC molecules, whereas peptides processed in the vesicular system (e.g., bacterial, viral) will vary in length from about 10 amino acids to about 25 amino acids and associate with class II MHC molecules. Since KMT2A::AFF1 is an internal host protein, KMT2A::AFF1 antigen peptides will be presented in the context of class I MHC. In particular aspects, 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. Thus, in some aspects, 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).

[0029] In certain embodiments, 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:1-2 and / or SEQ ID NOs:81-84 as presented herein, provided thatthe 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, 2ndEdition; 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. Alternatively, 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).

[0030] As used herein, "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). In certain embodiments, 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.

[0031] Vαriants 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.fthe amino acid sequence of RIRVDFKQTYSNEVH (SEQ ID NO:85). Accordingly, in some aspects, 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 β-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) .

[0032] "Sequence identity," as used herein, 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.

[0033] In some aspects, the recombinant TCR of the invention, or the antigen binding fragment thereof, is chimeric, humanized, or human. In some aspects, 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. In some aspects, a transmembrane domain allows for incorporation of an artificial TCR into a native TCR complex. In some aspects, 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. In some aspects, an endodomain includes at least one, two or three ITAMs. In some aspects, an endodomain includes a CD28 intracellular domain, an 0X40 intracellular domain, a CD3--zeta intracellular domain, or a chimeric intracellular domain thereof.

[0034] In some aspects, 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, by way of example, 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 recombinantpolypeptide. 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.

[0035] Certain aspects relate to isolated polynucleotides that encode the α-chain variable domain and / or β-chain variable domain of the TCR described herein. As used 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. In certain aspects, 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., α-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.

[0036] 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. As one of skill in the art will recognize, 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). For example, a 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. Such 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.

[0038] In some aspects, the invention provides an isolated polynucleotide encoding a recombinant T cell receptor, or antigen binding fragment thereof, including an α-chain variable domain having a CDR3 amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and / or a β-chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2). In some aspects, the polynucleotide further encodes an α-chain constant domain and / or a β-chain constant domain as described herein. In some aspects, the α-chain variable domainand β-chain variable domain (and optional constant chains) of the TCR are encoded by separate nucleic acid molecules. In some aspects, the a-chain variable domain and β-chain variable domain (and optional constant chains) of the TCR are encoded by the same nucleic acid molecule. In accordance with some aspects, a nucleic acid molecule encoding both the a-chain variable domain and β-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 β-chain variable domain.

[0039] In some aspects, the polynucleotides of the invention are provided in a vector. One of skill in the art can readily ascertain suitable vectors for use with certain aspects disclosed herein (e.g., cloning, expression, transfection, etc.). A typical 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. Some examples of 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"). According to some aspects, it is further understood that, if one or more agents (e.g., polynucleotides encoding recombinant TCRs specific for KMT2A: :AFF1, or variants thereof, as described herein) are co- administered to a subject (or host cell thereof), that eachagent 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.

[0040] As used herein, "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. Such 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. In certain aspects, a polynucleotide is codon optimized for efficient expression in a target host cell. In the present invention, "plasmid," "expression plasmid," "virus" and "vector" are often used interchangeably.

[0041] The term "expression", as used herein, 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.

[0042] In some aspects, 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) . Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus , and hepatitis virus, for example. Examples of 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). In some aspects, the viral vector is a lentiviral vector or a retroviral vector.

[0043] "Lentiviral vector, " as used herein, 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.

[0044] In certain aspects, the polynucleotide encoding the recombinant TCR specific for KMT2A::AFF1, may be operatively linked to certain elements of a vector. For example,polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are Ligated may be operatively linked. The term "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. For example, 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.

[0045] 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). To obtain efficient transcription and translation, a polynucleotide ina 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.

[0046] In some aspects, the polynucleotide sequence encoding the TCR or recombinant expression vector comprising the same is delivered to an appropriate cell, for example, a T cell. In some aspects, 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).

[0047] 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). One of skill in the art readily understands that many suitable host cells are available in the art. A host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids and / or proteins, as well as any progeny cells. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. 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 calciumphosphate 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).

[0048] In certain aspects, 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. In some aspect, 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. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2011 / 0243972; US2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), such that adaptation of these methodologies to the present invention is contemplated.

[0049] 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).

[0050] As used herein, 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). In certain aspects, a host cell mayoptionally 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). As used herein, 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. In certain embodiments, 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. In addition, "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.

[0051] As described herein, more than one 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. For example, as disclosed herein, a host cell may be modified to express two or more heterologous or exogenous nucleic acid molecules encodingdesired TCR specific for a KMT2A::AFF1 neoantigen (e.g., TCRα and TCRβ). When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that 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.

[0052] In some aspects, 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 β-chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2). In some aspects, 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. As used herein, the term '''endogenous" or "native" refers to a gene, protein, or activity that is normally present in a host cell.

[0053] Gene-editing nucleases may be employed in order to disrupt components of the endogenous TCR. Since the TCRα / β dimer can produce a fully functioning TCR complex, disrupting TCRα and / or TCRβ function may reduce (even eliminate) endogenous TCR expression. Vαrious methods may be used to disrupt endogenous TCRα or TCRβ 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 fingernucleases (ZFN) are heterodimeric arrays that co-localize at a target DNA site. ZFNs include individual finger subunits that bind DNA and are tethered to the Fokl nuclease domain that cleaves DNA. Transcription activator-like effector nucleases (TALEN) 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 (MN) 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. Any one or more of these gene editing enzymes may be used to disrupt expression of an endogenous TCRα and / or TCRβ to disrupt assembly of the endogenous TCRα and TCRβ.

[0054] In certain aspects, 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 TCRα chain and / or a TCRβ chain specific for a KMT2A::AFF1 antigen or antigen binding fragment thereof. As used herein, 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 CD24LoLin CD117+ phenotype or those found in the thymus (referred to as progenitor thymocytes).

[0055] As used herein, 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). 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. In some aspects, the immune system cell is a T cell, a natural killer cell, or a combination thereof.

[0056] A "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 (TM) (antigen-experienced and long-lived); and effector cells (antigen-experienced, cytotoxic). TMmay be further divided into subsets of central memory T cells (TCM, 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 (TEM, which exhibit decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or TCM). Effector T cells (TE) refer to antigen- experienced CD8+cytotoxic T lymphocytes that have decreasedexpression of CD62L, CCR7, CD28, and are positive for granzyrae and perforin as compared to TCM. 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.

[0057] T cells, or a subset of 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. 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.

[0058] 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. In some aspects, the T cells may be cultured under conditions effective for expanding the population of T cells.

[0059] 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. For example, CD3+, CD28+T cells can be positively selected using CD3 / CD28-conjugated magnetic beads. In one aspect of the present invention, 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.

[0060] In some situations, 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.

[0061] For example, 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. For example, cells may be washed to remove the plasma fraction. For example, T cells may be isolated from peripheral blood lymphocytes by lysing the red blood cells and by centrifugation through a PERCOLL™ gradient. Nucleic acids encoding the recombinant TCR or vectors containing such nucleic acids may subsequently be delivered to the isolated Tcells for expression and processing. Recombinant T cells expressing the TCR may be cultured under conditions effective for expanding the population of T cells.

[0062] In some aspects, the invention also provides methods for treating a hyperproliferative disorder or a condition characterized by KMT2A::AFF1 neoantigen expression. In particular, 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. As understood by a person skilled in the medical art, 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). In one aspect, 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 asubject 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 .

[0063] 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.

[0064] In certain aspects, 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.

[0065] 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. In some cases, a subject may be a human. In some cases, a subject may be a child (i.e., a young human being below the age of puberty). In some cases, a subject may be an infant. In some cases, the subject may be a formula-fed infant. In some cases, a subject may be an individual enrolled in a clinical study. In some cases, a subject may be a laboratory animal, for example, a mammal, or a rodent. In some cases, the subject may be a mouse.

[0066] In some aspects, 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 .

[0067] 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.

[0068] 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 significantmanner) in a treated human or non-human mammal. As is well known in the medical arts, 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. Doses will vary, but a preferred dose for administration of a host cell comprising a recombinant expression vector as described herein is about 107cells / m2, about 5*107cells / m2, about 108cells / m2, about 5x108cells / m2, about 109cells / m2, about 5×109cells / m2, about 1010cells / m2, about 5×1010cells / m2, or about 1011cells / m2.

[0069] Pharmaceutical 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. In general, 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). For prophylactic use, 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.

[0070] 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. For example, such 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. In some aspects, the host cells are expanded before and / or after introduction of the desired nucleic acid molecule .

[0071] As used herein, 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.

[0072] In some aspects, the host cell is co-administered with an adjunctive therapy (e.g., a cytokine such as IFN-γ, 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 hostcells 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). In other aspects, 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.

[0073] 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.

[0074] 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).

[0075] 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 response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.

[0076] 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.

[0077] The pharmaceutical 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. In certain aspects, a unit dose includes a recombinant host cell asdescribed herein at a dose of about 107cells / m2to about 1011cells / m2. The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including, e.g., parenteral or intravenous administration or formulation.

[0078] If the subject composition is administered parenterally, 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. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. Dosage unit form, as used herein, 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.

[0079] In general, an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit. Such 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 withan 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.

[0080] The following non-limiting examples are provided to further illustrate the present invention.Example 1: Materials and Methods

[0081] 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.

[0082] TABLE 1a, relapse disease stage;b, diagnosis disease stage.TABLE 2a, relapse disease stage;b, diagnosis disease stage.

[0083] Tn Vitro T-Cell Expansion. 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 mediacontaining IL2 every 2 days. At Day 15, the expanded T cells were either used in co-culture assays or cryopreserved.

[0084] Assessment of T-Cell Reactivity: IFN-y ELISPOT Assay and Detection of Activation Markers by Flow Cytometry. T-cell reactivity was assessed by IFN-γ 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-γ secretion was measured by ELISPOT. Before the ELISPOT assay was processed, 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.

[0085] Single-Cell TCR Sequencing. The 4-1BB+ CD8+T cells and 4-lBB+ / OX40+CD4+T cells were individually sorted into 384- well plates, and TCRα and TCRβ chains were amplified by nestedPCR using variable and constant region-specific primers. A modification to this protocol was the inclusion of well barcodes in second-round primers. PCR products from all wells on a plate were pooled 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.

[0086] 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- Prkdcscid·I12rgtm1WjlTg(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 .

[0087] Ex vivo Culture of ALL, AML, MPAL Blasts. Prior to T- cell reactivity evaluation, leukemia blasts from all patients were initially expanded by co-culture with mesenchymal stem cells, as previously described (Pal et al. (2016) Leukemia 30(8) :1691-1700). In brief, mesenchymal stem cells were seeded at a concentration of 104cells / cm2in RPMI supplemented with 20% fetal bovine serum (FBS) and 1 μm hydrocortisone 48 hours before adding leukemia cells. The media was aspirated, and leukemia cells were added at a concentration of 1-2 x 106cells / mL in SFEM II media. Culture media were supplementedwith 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.

[0088] Generation of 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) . Cells were expanded for 5 days and re- stimulated for 3 rounds in B-cell media; Iscove's Modified Dulbecco's Medium (IMDM; ATCC) containing 10% human serum AB, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, and 200 U / mL IL4 (Peprotech). Following the expansion, the cells were either used in co-culture assay or cryopreserved. When 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).

[0089] Flow Cytometry and Cell Sorting. Cells were subjected to FACS analysis by incubation with a mix of fluorescently labeled antibodies for 30 minutes at 4°C after blocking Fc receptors with Human TruStain FcX™ solution (BioLegend) for 10 minutes at 4°C. Flow cytometry analysis was performed using a Cytek Aurora spectral cytometer. Cells were sorted using a Bigfoot spectral cell sorter (ThermoFisher). Data were analyzed using FlowJo software.

[0090] 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. (2018) JCI Insight 3(19):el22467). Single cells were sorted into a 384-well plateand 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.

[0091] 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).

[0092] CRISPR-Cas9 TRAC Knockout. 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 μg / 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 × 106cells / mL and activated for 48 hours. Following incubation, the beads were removed, washed, and resuspended in Buffer P3 Primary Cell Nucleofector (Lonza) at a concentration of 5-10 × 104cells / mL in 100-μL volume. CRISPR-Cas9 ribonucleoproteins, formulated by complexing 6 μM Cas9 and 6 μM sgTRAC (1:1 ratio), were added to the cells and then electroporated (Lonza, X-unit, EH-115). The cells were recovered by adding RPMI supplemented×ith 10% FBS and 180 lU / mL IL2 at a concentration of 2 × 106cells / mL for 16-24 hours before TCR retrovirus transduction.

[0093] 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 μg expression plasmid, 9 μg Peq-Pam, 4 μg RD114, and 60 μL 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.

[0094] Construction of Minigene, in vitro Transcription, and Electroporation, Fusion minigenes were constructed by including 100 amino acids flanking the breakpoint and cloned into the pcDNA3.1 vector. The sequences were verified by Sanger sequencing. The vector was linearized and then purified using NUCLEOSPIN® Gel and PCR Clean-up (Macherey-Nagel). HISCRIBE® T7 ARCA mRNA Kit, with tailing (NEB) was used to generate in vitro transcription RNA per the manufacturer's instructions. RNA was purified by LiCl2precipitation and resuspended in H2O at a concentration of 1 μg / μL.

[0095] B cells were washed and resuspended at a concentration of 5-10 x 106cells in 100 μL optiMEM™. Then 8 μg 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 GenePulser. The B cells were incubated at 37°C, under 5% CO2, for 16 hours and used for co-culture assays.

[0096] 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 × 104cells) were stained with 0.25 pM CFSE by incubating at 37°C, under 5% CO2, 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. Following incubation, the cells were stained with Live / Dead Ghost Dye™ Violet 510 (Tonbo) for 30 minutes at room temperature. The cells were then washed and resuspended in staining buffer containing 5 μL CountBright™ 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 μg / mL blasticidin was used as the positive control.

[0097] 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.

[0098] Gene fragments encoding patients' HLA were acquired from Twist Bioscience. Each HLA sequence was cloned into the μLVX-EFla-IRES-Puro lentivirus expression vector (Clonetech). Lentivirus was produced by co-transfection of pLVX lentivirus vector containing an HLA insert, psPAX2 packaging plasmid, andpMD2.G envelope plasmid into the 293T packaging cell line by using polyethylenimine (PEI). The cells were washed 4 hours after co-transfection. After 24 and 48 hours, the viral supernatant was collected and filtered, K562 cells expressing MHC class II machineries were subsequently transduced and subjected to puromycin selection for 1 week in IMDM supplemented with 10% FBS. The expression of single HLA molecules was verified by flow cytometry.

[0099] Bulk TCR Library Prep and Sequencing. TCRα and TCRβ 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). RNA was extracted and reverse- transcribed using SmartScribe™ RT reagent (Takara), and Q5 polymerase (New England Biolabs) was used during first- and second-round amplifications. Barcoded TCRα and TCRβ 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.

[0100] Processing Bulk and Single-Cell TCR Sequencing. MIXCR (V4.2.0; Bolotin et al. (2015) Nat. Methods. 12 (5):380-381) using the analyze amplicon routine was used for demultiplexing, alignment, and clonotype assembly beginning from paired-end FASTQ reads using the analyze generic-tcr- amplicon-separate-samples-umi and generic-tcr-amplicon built- ins for bulk and single-cell processing, respectively. Custom R (v4.1.2) scripts were used to determine paired chains from parsed single-cell TCR data.Example 2: T-Cell Expansion in Leukemias Characterized by Genetic Fusions

[0101] 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. (2018) Nature 562(7727):373-379) (Table 2). The compositions of bone marrow and peripheral blood were dominated by leukemic blasts (median, 92%; range, 59%-99%), and the median frequency of lymphocytes was only 3.5% (range, l%-25%, Tables 1-2). To adequately assess T cell reactivity, the number of T cells were expanded using a rapid expansion protocol (REP) and cultured for 14 days to overcome the low lymphocyte cellularity. Amplification was successful in all samples, irrespective of genetic function or lymphocyte count. 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. The median frequencies of CD4+and CD8+T cells after the expansion, compared with that of CD3+T cells, were 59.7% and 26.1%, respectively.Example 3: T-Cell Recognition And Reactivity Against Autologous ALL Blasts

[0102] It was examined whether the expanded T cells recognize and react to autologous leukemic blasts by measuring IFN-γ secretion and upregulation of co-stimulatory receptors 4-1BBand 0X40 after overnight co-culture, as previously reported (Tran et al. (2015) Science 350 (6266):1387-1390; Gros et al. (2019) J. Clin. Invest. 129(11):4992-5004). An increase in 4- 1BB expression on both CD8+and CD4+T cells was observed in 13 of 15 ALL samples. Moreover, 0X40 was highly upregulated on CD4+cells and IFN-γ secretion was increased, as determined by ELISPOT assay. Although upregulation of these activation markers was not observed in one sample (SJMLL009), IFN-γ secretion in that sample was markedly increased compared to that in the irrelevant control (119 spots in blast co-culture, 7 spots in irrelevant control, and 0 spots in T cells only control) .

[0103] Of the 15 ALL samples, only SJALL048347 did not show a T-cell response. It was posited that the blasts in that sample might have lower MHC expression or antigen presentation that could be induced by IFN-γ exposure, as previously reported (Christopher et al. (2018) New England Journal of Medicine 379(24):2330-2341; Puig-Saus et al. (2023) Nature 615 (7953):697-704). To that end, leukemic blasts were pre- treated with 50 ng / mL of IFN-γ for 72 hours, washed, and then co-cultured with autologous T cells. T-cell reactivity was remarkably increased, as indicated by the upregulation of 4- 1BB and IFN-γ secretion. This response was associated with a simultaneous 2.6~fold and 1.8-fold upregulation in MHC class I and II, respectively.

[0104] To further expand on these findings, 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 demonstratedeffective T-cell mediated killing of autologous leukemic blasts (FIG. 1), indicating T-cell reactivity in all 15 patients with ALL.Example 4: T-Cell Recognition and Reactivity Against Autologous AML and MPAL Blasts

[0105] 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. Unlike the ALL blasts, the AML and MPAL blasts required IFN-γ treatment prior to co-culturing. Without the pre- exposure to IFN-γ, most samples showed either minimal or no T-cell reactivity. MHC class I and II expression after IFN-γ 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.

[0106] The expanded T cells were co-cultured with their autologous IFN-γ 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-γ production and 0X40 expression by CD4+T cells. Like the ALL sample SJMLL009, the AML sample SJMLL012 showed T-cell reactivity by IFN-γ secretion and 0X40 on CD4+T cells, despite no upregulation of the 4-1BB marker. Of the 15 AML samples, only SJAML061496 showed no reactivity. All 4 MPAL samples showed upregulation of 4-1BB and 0X40 and increased IFN-γ secretion.

[0107] 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

[0108] Recent work in solid tumors, including breast, head and neck, endometrial, and non-small cell lung cancer, shows that PD1 and CD39 mark tumor-reactive T cells. Furthermore, the expression of PD1hiand CD39 can be used to specifically select and expand tumor-reactive T cells. Thus, it was investigated whether these markers could also identify leukemia-reactive T cells.Three samples obtained at diagnosis, including ALL with KMT2A: :MLLT10 (SJALL016500), AML with PICALM: :MLLT10 (SJAML030459), and AML with NUP98::NSD1 (SJAMLOO1441) were evaluated. All samples included a PD1+CD39+population of T cells, but it was smaller in SJAML001441. T cells were isolated based on the markers they expressed (i.e., PD1- CD39-, PD1~ CD39+, and PDlhi) and then expanded in vitro. The expanded T cells were then co-cultured with autologous blasts to assess their reactivity. PD1~ CD39+ and PDhipopulations had up to 2.5-fold greater response to leukemia blasts, as shown by 4- IBB upregulation and increased IFN-γ secretion. Furthermore, the PDlhipopulation co-expressed CD39 and TIM3 at higher levels than did the PDldimor 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 recognizedas 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.Example 6: Leukemia Blast- and Fusion Gene-Reactive T-Cell Receptors

[0109] To identify the specific T cell receptors (TCRs) that recognize leukemic blasts and fusion gene-derived neoantigens, 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. Importantly, infant ALL with W T2A-rearrangements has a mean of 1.3 non-silent mutations, which lowers the complexity of the neoantigen screening.

[0110] Single-cell TCR sequencing of the sorted populations showed the presence of major clonotypes in 4 (SJINF002, SJINF013, SJAML030459, SJAML001441) of the 5 samples (Tables 3-4) .TABLE 3TABLE 4

[0111] 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. (2018) 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. Fusion genes were overexpressed on their corresponding antigen-presenting cells (APCs) and co-cultured with peripheral blood lymphocytes containing the candidate TCRs. 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). Despite not showing reactivity to the fusion neoantigen, the most prevalent TCR clonotype of SJINF013 and SJAML001441 showed reactivity to leukemic blasts. Furthermore, sample SJAML030471, which did not have any major clonotypes, lacked TCR reactivity to both fusion neoantigen and leukemic blasts.

[0112] 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). Notably, the most prevalent 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.

[0113] Subsequently, the human leukocyte antigen (HLA)- restricted elements that presented the fusion neoantigens were examined. HLA typing was performed (Table 5), and individual MHC II alleles were transiently transfected into the 293T- CIITA cell line with fusion gene RNA.TABLE 5

[0114] Sequencing identified the KMT2A::AFF1 from patient SJINF002 with the breakpoint located at exons 10 and 4, respectively. This fusion was recognized by TCR SJINF002 clone 2 when presented by DPAl*02:01 / DPBl*01:01 (FIG. 5A). This result was confirmed using the K562 artificial antigen presenting cell model as an alternative antigen-presentation source (Zamora et al. (2019) Sci. Transl. Med. 11(498) :eaat8549). The K562 cell line was transduced with MHC class II machinery, including CD64, CD80, CD83, CD74, and HLA- DM, using a lentivirus vector. Individual MHC allele, fusion gene, or wild-type control sequences were transfected into the cell line and maintained in co-culture with the candidate TCR. Consistent results of TCR reactivity to the KMT2A::AFF1 fusion neoantigen were observed when presented by DPAl*02 :01 / DPBl*01:01.

[0115] 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-γ secretion (FIGS. 5B-5C).

[0116] Subsequently, minimal epitope prediction was performed on the fusion neoantigen KMT2A-.:AFF1 when presented by HLA- DPA1*O2:01 / DPBl*01:01 using NetMHCIIpan 4.0 (Reynisson et al. (2020) Nucleic Acids Res. 48(W1):W449-W454). The peptide was synthesized and pulsed into K562 MHC II cells containing this HLA allele. Activation marker upregulation and increased IFN- y secretion of T cells expressing TCR SJINF002 clone 2 to the fusion peptide RIRVDFKQTYSNEVH (SEQ ID NO:85) indicated recognition of the fusion (FIG. 6). The abundance of this clonotype across time points, including at diagnosis (Day 0), first remission (Day 198), relapse (Day 1006), and remission after relapse (Day 2238), was tracked. 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. Leukemia-reactive clonotypes longitudinally in samples SJINF013 and SJAML030459 were evaluated. Similarly, they were only found at low frequencies and only at the diagnostic timepoint, including 0.04% (SJINF013 clone 2), 0.05% (SJAML030459 clone 3), and 0.4% (SJAML030459 clone 6).Example 7: TCR SJINF002_2-expressing T Cells Reduce Tumor Burden and Improve Survival

[0117] NSG 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. 7A) and expansion of the SJINF002_2 TCR T cells (FIG. 7B) were observed. This reduction correlated with significantly higher body weight (FIG. 7C) and improved survival (FIG. 7D) in the SJINF002_2~treated mice compared to controls. Additionally, SJINF0022 TCR T cells remained detectable in the bone marrow up to the study endpoint (approximately 4500 T cells per bone marrow of SJINF002_2 TCR T cells compared to 400 cells per bone marrow of the irrelevant control TCR T cells).

Claims

WHAT IS CLAIMED IS:

1. A recombinant T cell receptor, or antigen binding fragment thereof, comprising an α-chain variable domain having a CDR3 amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and / or a β-chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID N0:2).

2. The recombinant T cell receptor of claim 1, wherein the T cell receptor is capable of binding to a KMT2A::AFF1 fusion neoantigen.

3. The recombinant T cell receptor of claim 1, further comprising an α-chain constant domain having at least about 90% sequence identity to an amino acid sequence of SEQ ID NO:81 or SEQ ID NO:82.

4. The recombinant T cell receptor of claim 1, further comprising a β-chain constant domain having at least about 90% sequence identity to an amino acid sequence of SEQ ID NO:83 or SEQ ID NO:84.

5. The recombinant T cell receptor of claim 1, wherein the T cell receptor, or the antigen binding fragment thereof, is chimeric, humanized, or human.

6. The recombinant T cell receptor of claim 1, wherein the antigen binding fragment of the T cell receptor comprises a single chain T cell receptor (scTCR).

7. An isolated polynucleotide encoding the recombinant T cell receptor of claim 1.

8. An expression vector comprising a polynucleotide encoding a recombinant T cell receptor, or antigen binding fragment thereof, comprising an α-chain variable domain having a CDR3 amino acid sequence of CSVRRNSNYQLIW (SEQ ID NO:1) and / or a β-chain variable domain having a CDR3 amino acid sequence of CSVVRGEGYEQYF (SEQ ID NO:2).

9. The expression vector of claim 8, wherein the polynucleotide encoding the recombinant T cell receptor, or antigen binding fragment thereof, is operably linked to an expression control sequence.

10. The expression vector of claim 8, wherein the vector is capable of delivering the polynucleotide to a host cell.

11. The expression vector of claim 8, wherein the vector is a viral vector.

12. The expression vector of claim 11, wherein the viral vector comprises a lentiviral vector or a retroviral vector.

13. A recombinant host cell comprising the isolated polynucleotide of claim 7, wherein the host cell expresses on its cell surface the recombinant T cell receptor encoded by the polynucleotide.

14. The recombinant host cell of claim 13, wherein the host cell is allogeneic, syngeneic, or autologous.

15. The recombinant host cell of claim 13,wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

16. The recombinant host cell of claim 15, wherein the immune system cell is a T'cell, a natural killer cell, or a combination thereof.

17. The recombinant host cell of claim 16, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

18. The recombinant host cell of claim 13, wherein the host cell has reduced expression or does not express an endogenous T cell receptor.

19. 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 comprising administering to the subject a recombinant host cell of claim 13 thereby treating the subject's leukemia.

20. The adoptive immunotherapy method of claim 19, wherein the recombinant host cell is modified ex vivo.

21. The adoptive immunotherapy method of claim 19,wherein the host cell is an allogeneic cell, a syngeneic cell, or an autologous cell.

22. The adoptive immunotherapy method of claim 19, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

23. The adoptive immunotherapy method of claim 22, wherein the immune system cell is a T cell, a natural killer cell, or a combination thereof.

24. The adoptive immunotherapy method of claim 23, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

25. The adoptive immunotherapy method of claim 19, wherein the host cell does not express or has reduced expression of an endogenous T cell receptor.

26. The adoptive immunotherapy method of claim 19, wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), or mixed phenotypic leukemia.

27. The adoptive immunotherapy method of claim 19, further comprising administering a cytokine concurrently or sequentially with the recombinant host cell.

28. The adoptive immunotherapy method of claim 27, wherein the cytokine is IFN-γ.

29. A unit dose form comprising a recombinant host cell of claim 13.

30. The unit dose form of claim 29, wherein the recombinant host cell is at a dose of about 107cells / m2to about 1011cells / m2.

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