Compositions and methods for immunotherapy of NPM1c-positive cancers

Abstract

The present disclosure relates to compounds (e.g., antibodies, antigen-binding fragments thereof, bispecific molecules, or chimeric antigen receptor polypeptides) that bind to a neoepitope of mutant nucleophosmin (NPM1c) complexed with or presented by class I major histocompatibility complex (MHC class I) proteins, or cells expressing such compounds, and their use in methods for treating or ameliorating one or more symptoms of cancer.

Description

[Technical Field]

[0001] Government license statement This invention was made with government support under Grant No. CA197605 awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 987,612, filed March 10, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Cell-based immunotherapy is currently being developed for the treatment of cancer. Approaches using adoptive cell transfer of T cells, monocyte-derived cells (e.g., macrophages, dendritic cells), and natural killer (NK) cells are being investigated as cancer treatments (see, e.g., Andreesen, R. et al. (1990) Cancer Res 50:7450-7456; Ruggeri, L. et al. (2002) Science 295:2097-2100; Rezvani, K. (2019) Bone Marrow Transplantation 54:785-788). Specifically, adoptive cell therapy (ACT), in which ex vivo activated / expanded T cells are administered to patients, is one cancer treatment currently being tested. (Rosenberg et al. (2008) Nat Rev Cancer 8(4): 299; Dudley et al. (2002) Science 298(5594): 850; June et al. (2007) J Clin Invest 117(5): 1204; Stephan et al. (2007) Nat Med 13(12): 1440; Yee et al. (2002) Proc Natl Acad Sci USA 99(25): 16168). These approaches involve the use of autologous T cells harvested from the patient, activated / expanded ex vivo, and then reinfused to fight tumors, e.g., metastatic tumors. Strategies to enhance the persistence, in vivo expansion, and effector function of ACT T cells have been used to increase the frequency of objective responses. (Rosenberg SA et al. (2008) Nat Rev Cancer 8(4): 299; June CH et al. (2007) J Clin Invest 117(5): 1204).One way to enhance the function of ACT T cells is through genetic manipulation of the cells themselves, for example, by introducing chimeric receptors or costimulatory molecules (see, e.g., Stephan et al. (2007) Nat Med 13(12): 1440; Morgan et al. (2006) Science 314(5796): 126; Gade et al. (2005) Cancer Res 65(19): 9080).

[0004] Chimeric antigen receptor (CAR) T cell therapy has emerged as a strategy for cancer treatment. Chimeric antigen receptors (CARs) are genetically engineered, artificial transmembrane receptors that confer defined specificity to an antigen (e.g., a ligand) to immune effector cells (e.g., T cells, natural killer cells, or other immune cells), resulting in activation of the effector cell upon recognition and binding to the antigen. Typically, these chimeric receptors are used to confer the antigen specificity of a monoclonal antibody to T cells, referred to in the art as CAR T cells. Expression of the engineered chimeric antigen receptor on the surface of CAR T cells confers the ability to lyse any target cell bearing surface expression of the specific antigen recognized by the chimeric receptor.

[0005] However, current CARs targeting lineage-restricted or tumor-associated antigens (TAAs) can be highly toxic due to low antigen expression in normal tissues (see Coulie et al., NAT REV CANCER 14: 135 (2014); Srivastava & Riddell, J IMMUNOL 200: 459 (2018)). Furthermore, because TAAs are not required for tumor cell survival, loss of TAA expression is a major cause of tumor resistance to CAR-T therapy (see Srivastava & Riddell, J IMMUNOL 200: 459 (2018)). Neoantigens are derived from tumor-specific gene mutations, and their formation and expression are restricted to malignant cells (see Blankenstein et al., CURR OPIN IMMUNOL 33 112 (2015); Schumacher et al. SCIENCE 348: 69 (2015); van der Lee et al., J CLIN INVEST 129: 774 (2019)). However, the majority of neoantigens are encoded by patient-specific passenger mutations that can be lost due to immunoediting, resulting in tumor immune evasion (see Verdegaal et al., NATURE 536: 91 (2016)). In addition, current CARs are primarily designed to bind to antigens on the surface of target cells. In reality, most proteins derived from mutant genes are expressed intracellularly and therefore unavailable as targets for conventional CARs (see Uhlen et al., SCIENCE 347: 1260419 (2015)).

[0006] Cancer immunotherapy has limited application in acute myeloid leukemia (AML), a rapidly progressing hematopoietic malignancy characterized by the accumulation of differentiation-arrested malignant myeloid progenitor cells in the bone marrow (see van der Lee et al., J CLIN INVEST 129: 774 (2019); Thomas et al., BLOOD 129: 1577 (2017)). The current standard of care for AML remains intensive chemotherapy and autologous or allogeneic hematopoietic stem cell transplantation (alloSCT) (see Dombret & Gardin, BLOOD 127: 53 (2016); Dohner et al., N Engl J Med 373: 1136 (2015)). Although most patients can achieve complete remission in response to standard treatment, approximately 50% of these patients will relapse (see Ossenkoppele et al., HAEMATOLOGICA 101 20 (2016)). Patients with relapsed or refractory AML after intensive chemotherapy or alloSCT usually have a very poor prognosis (see van der Lee et al., J CLIN INVEST 129: 774 (2019)). Therefore, there is a great demand for developing new, effective, and less toxic treatments for these patients. Summary of the Invention

[0007] In some aspects, the present disclosure provides antibodies, or antigen-binding fragments thereof, that specifically bind to an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein.

[0008] In some aspects, the antibody or antigen-binding fragment thereof does not bind or does not substantially bind to (a) an MHC class I protein alone and / or (b) a control peptide complexed with an MHC class I protein, and optionally the control peptide is an NY-ESO-1 epitope or an influenza virus M1 epitope.

[0009] In some aspects, the antibody or antigen-binding fragment thereof does not bind or does not substantially bind to (a) an MHC class I protein alone and (b) a control peptide complexed with an MHC class I protein, and optionally the control peptide is an NY-ESO-1 epitope or an influenza virus M1 epitope.

[0010] In some aspects, the antibody, or antigen-binding fragment thereof, does not bind or does not substantially bind to (a) an MHC class I protein alone, (b) a control peptide complexed with an MHC class I protein, optionally the control peptide being an NY-ESO-1 epitope or an influenza virus M1 epitope, and / or (c) an NPM1c neoepitope alone.

[0011] In some aspects, the antibody, or antigen-binding fragment thereof, does not bind or does not substantially bind to (a) an MHC class I protein alone, (b) a control peptide complexed with an MHC class I protein, optionally the control peptide being an NY-ESO-1 epitope or an influenza virus M1 epitope, and (c) an NPM1c neoepitope alone.

[0012] In any of the above or related embodiments, the NPM1c neoepitope comprises the amino acid sequence X1X2X3X4X5X6X7X8X9, where X1 is selected from A, V, L, or I; X2 is selected from A, T, S, V, L, I, M, or Q; X3 is selected from Q or N; X4 is selected from D or E; X5 is selected from L, I, V, M, A, or F; X6 is selected from C, S, or A; X7 is selected from L, I, V, M, A, or F; X8 is selected from A, V, L, or I; and X9 is selected from L, I, V, M, or A. In some embodiments, the NPM1c neoepitope comprises the amino acid sequence X1X2X3X4X5X6X7X8X9, where X1 is selected from A or V, X2 is selected from V, I, or L, X3 is selected from Q or N, X4 is selected from D or E, X5 is selected from L or I, X6 is selected from C or S, X7 is selected from V, L, or I, X8 is selected from A or V, and X9 is selected from V, I, or L. In some embodiments, the NPM1c neoepitope comprises the amino acid sequence X1X2X3X4X5X6X7X8X9, where X1 is A, X2 is selected from V, I, or L, X3 is Q, X4 is D, X5 is L, X6 is C, X7 is L, X8 is A, and X9 is selected from V, I, or L. In some embodiments, the NPM1c neoepitope is within a peptide that is 10, 15, 20, 30, 40, 50, or 100 amino acid residues in length.

[0013] In any of the above or related embodiments, the NPM1c neoepitope comprises an amino acid sequence selected from AIQDLCLAV (SEQ ID NO: 1) or AIQDLCVAV (SEQ ID NO: 71). In some embodiments, the NPM1c neoepitope comprises an amino acid sequence selected from CLAVEEVSL (SEQ ID NO: 72), VEEVSLRK (SEQ ID NO: 73), AVEEVSLR (SEQ ID NO: 74), AVEEVSLRK (SEQ ID NO: 75), CLAVEEVSLRK (SEQ ID NO: 76). In some embodiments, the NPM1c neoepitope comprises the amino acid sequence AIQDLCLAV (SEQ ID NO: 1).

[0014] In any of the above or related embodiments, the NPM1c neoepitope is 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues in length.

[0015] In any of the above or related embodiments, the MHC class I protein is an HLA-A*02 protein or is encoded by the HLA-A*02 allele group. In some embodiments, the MHC class I protein is encoded by the HLA-A*02:01 allele.

[0016] In any of the above or related aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising: (i) a heavy chain variable region (VH) comprising a VH complementarity-determining region (CDR) 1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 are the CDRs of a VH having the amino acid sequence of SEQ ID NO: 5, and the CDRs are as defined by IMGT; and / or (ii) a light chain variable region (VL) comprising a complementarity-determining region (CDR) 1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 are CDRs of a VL having the amino acid sequence of SEQ ID NO: 3, and the CDRs are as defined by IMGT; The present invention provides an antibody, or antigen-binding fragment thereof, comprising:

[0017] In any of the above or related aspects, the disclosure provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising a complementarity determining region (CDR) 1, a VH CDR2, and a VH CDR3, wherein VH CDR1 has the amino acid sequence GFTFSSYA (SEQ ID NO: 9), VH CDR2 has the amino acid sequence ISGSGGST (SEQ ID NO: 10), and VH CDR3 has the amino acid sequence ARLGYPTTTLLPFDY (SEQ ID NO: 11).

[0018] In any of the above or related aspects, the disclosure provides an antibody, or antigen-binding fragment thereof, further comprising a light chain variable region (VL) comprising a VL complementarity determining region (CDR) 1, a VL CDR2, and a VL CDR3, wherein VL CDR1 has the amino acid sequence QSISSY (SEQ ID NO: 6), VL CD2 has the amino acid sequence AAS (SEQ ID NO: 7), and VL CD3 has the amino acid sequence QQSYSTPLT (SEQ ID NO: 8).

[0019] In any of the above or related aspects, the disclosure provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence at least 90% identical, or at least 95% identical, to the amino acid sequence of SEQ ID NO:5, and / or the VL comprises an amino acid sequence at least 90% identical, or at least 95% identical, to the amino acid sequence of SEQ ID NO:3.

[0020] In any of the above or related aspects, the disclosure provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO:5 and / or the VL comprises the amino acid sequence of SEQ ID NO:3.

[0021] In any of the above or related aspects, the disclosure provides an antibody, or antigen-binding fragment thereof, that is a human antibody, a humanized antibody, or a chimeric antibody.

[0022] In any of the above or related aspects, the disclosure provides an antibody, or antigen-binding fragment thereof, that is a single-chain Fv (scFv), an Fv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single-chain antibody molecule.

[0023] In any of the above or related embodiments, the disclosure provides an antibody, or antigen-binding fragment thereof, that is an scFv. In some embodiments, the scFv is a human scFv. In some embodiments, the scFv comprises a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is a Gly-Ser linker. In some embodiments, the Gly-Ser linker is selected from the group consisting of (Gly4Ser) (SEQ ID NO: 58), (Gly4Ser) (SEQ ID NO: 59), (Gly4Ser) (SEQ ID NO: 60), and (Gly4Ser) (SEQ ID NO: 61). In some embodiments, the Gly-Ser linker comprises the amino acid sequence SGSSGGSSSG (SEQ ID NO: 4).

[0024] In any of the foregoing or related embodiments, the scFv has an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence of SEQ ID NO:2, and optionally the scFv comprises: (a) a heavy chain variable region (VH) comprising a VH complementarity determining region (CDR) 1, a VH CDR2, and a VH CDR3, wherein VH CDR1 has the amino acid sequence GFTFSSYA (SEQ ID NO:9), VH CDR2 has the amino acid sequence ISGSGGST (SEQ ID NO:10), and VH CDR3 has the amino acid sequence ARLGYPTTTLLPFDY (SEQ ID NO:11); and / or (b) a light chain variable region (VL) comprising a VL CDR) 1, a VL CDR2, and a VL CDR3, wherein VL CDR1 has the amino acid sequence QSISSY (SEQ ID NO:6), VL CDR2 has the amino acid sequence AAS (SEQ ID NO:7), and VL CDR3 has the amino acid sequence AAS (SEQ ID NO:8). CD3 comprises a VL having the amino acid sequence QQSYSTPLT (SEQ ID NO: 8).

[0025] In any of the above or related embodiments, the scFv has the amino acid sequence of SEQ ID NO:2.

[0026] In any of the above or related aspects, the present disclosure provides an antibody, or antigen-binding fragment thereof, that is an antibody. In some aspects, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody isotypes. In some aspects, the antibody is of the IgG1 or IgG4 isotype. In some aspects, the antibody comprises a wild-type IgG1 heavy chain constant region or a wild-type IgG4 heavy chain constant region. In some aspects, the antibody comprises a mutant IgG1 heavy chain constant region or a mutant IgG4 heavy chain constant region.

[0027] In any of the above or related embodiments, the antibody comprises a mutated IgG4 heavy chain constant region, wherein the mutated IgG4 heavy chain constant region comprises any one of the following substitutions: S228P, L235E, L235A according to EU numbering, or a combination thereof.

[0028] In any of the above or related embodiments, the antibody comprises an Fc domain comprising at least one mutation.

[0029] In any of the above or related embodiments, the antigen is on the surface of a cancer cell. hi some embodiments, the cancer is acute myeloid leukemia (AML).

[0030] In any of the above or related embodiments, the antibody, or antigen-binding fragment thereof, binds to an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein with an equilibrium dissociation constant (Kd) of 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 0.5 nM to 100 nM, or 1 nM to 15 nM.

[0031] In any of the above or related embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that is a bispecific antibody, or antigen-binding fragment thereof, that further specifically binds to a second antigen on an immune effector cell. In some embodiments, the effector cell is a T cell, a natural killer cell, or a macrophage. In some embodiments, the second antigen is CD3. In some embodiments, the CD3 is human CD3 expressed on a T cell. In some embodiments, the second antigen is NKp46. In some embodiments, the NKp46 is human NKp46 expressed on an NK cell. In some embodiments, the second antigen is CD16A. In some embodiments, the CD16A is human CD16A expressed on an NK cell. In some embodiments, the second antigen is CD40, CD47, 4-1BB, TGF-β, LAG-3, PD-1, TIM-3, CTLA-4, OX40, NKp30, NKG2A, NKG2D, or DNAM-1.

[0032] In any of the above or related aspects, the disclosure provides a purified antibody, or antigen-binding fragment thereof.

[0033] In some aspects, the present disclosure provides an isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding an antibody, or antigen-binding fragment thereof, described herein.

[0034] In any of the above or related embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the isolated nucleic acid comprises a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 12.

[0035] In some aspects, the present disclosure provides an expression vector comprising a nucleic acid described herein. In some aspects, the present disclosure provides a cell transformed with an expression vector comprising a nucleic acid described herein.

[0036] In some aspects, the present disclosure provides a method for producing an antibody, or antigen-binding fragment thereof, described herein, comprising maintaining a cell transformed with an expression vector comprising a nucleic acid described herein under conditions that allow expression of the antibody, or antigen-binding fragment thereof. In some aspects, the method further comprises purifying the antibody, or antigen-binding fragment thereof.

[0037] In some aspects, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, described herein and a pharmaceutically acceptable carrier.

[0038] In some aspects, the present disclosure provides a chimeric antigen receptor (CAR) polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular binding domain, wherein the extracellular binding domain specifically binds to an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein.

[0039] In some aspects, the present disclosure provides a chimeric antigen receptor (CAR) polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular binding domain, wherein the extracellular binding domain comprises an antibody described herein, or an antigen-binding fragment thereof.

[0040] In any of the above or related embodiments, the transmembrane domain comprises the transmembrane domain of CD3-zeta, CD8, CD28, NKG2D, CD16, NKp44, or NKp46. In some embodiments, the intracellular domain comprises one or more costimulatory domains of one or more costimulatory molecules selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, 2B4, DAP10, CD137, and DAP12.

[0041] In any of the above or related aspects, the present disclosure provides a CAR polypeptide, wherein the intracellular domain comprises a CD3-zeta signaling domain and a 4-1BB costimulatory domain, the transmembrane domain comprises a CD8 transmembrane domain, and the CAR polypeptide further comprises a CD8 hinge region.

[0042] In any of the above or related aspects, the disclosure provides a CAR polypeptide, wherein the intracellular domain comprises a CD3-zeta signaling domain comprising the amino acid sequence set forth in SEQ ID NO:27, and a 4-1BB costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:26; the CAR polypeptide comprises a CD8 transmembrane domain and a CD8 hinge region, wherein the CD8 transmembrane domain and the CD8 hinge region comprise the amino acid sequence set forth in SEQ ID NO:25; and the extracellular binding domain comprises an antibody, or antigen-binding fragment thereof, and a leading sequence comprising the amino acid sequence set forth in SEQ ID NO:23.

[0043] In any of the above or related embodiments, the antibody or antigen-binding fragment thereof within the extracellular binding domain is an scFv comprising the amino acid sequence set forth in SEQ ID NO:24, or an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence of SEQ ID NO:24.

[0044] In any of the above or related embodiments, the intracellular domain further comprises a self-cleaving peptide sequence and a cytokine, wherein cleavage of the self-cleaving peptide releases the cytokine. In some embodiments, the cytokine is IL-12, IL-7, IL-13, IL-15, TNF-α, IFN-γ, or CCL19.

[0045] In any of the above or related aspects, the disclosure provides a CAR polypeptide comprising the amino acid sequence set forth in SEQ ID NO:22, or an amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence of SEQ ID NO:22.

[0046] In some aspects, the present disclosure provides an isolated nucleic acid encoding a CAR polypeptide described herein. In some aspects, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the nucleotide sequence of SEQ ID NO: 30. In some aspects, the present disclosure provides an expression vector comprising an isolated nucleic acid encoding a CAR polypeptide described herein, wherein the expression vector is a viral expression vector or a non-viral expression vector. In some aspects, the present disclosure provides an expression vector comprising an isolated nucleic acid encoding a CAR polypeptide described herein, wherein the expression vector is a viral expression vector, and wherein the viral expression vector is a lentiviral expression vector.

[0047] In some aspects, the present disclosure provides a cell transformed with an expression vector comprising an isolated nucleic acid encoding a CAR polypeptide described herein. In some aspects, the present disclosure provides a cell expressing a CAR polypeptide described herein. In some aspects, the cell is an immune effector cell, and expression of the CAR polypeptide causes the immune effector cell to target cancer cells expressing an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein. In some aspects, the MHC class I protein is an HLA-A*2 protein or is encoded by the HLA-A*02 allele group. In some aspects, the immune effector cell does not substantially target and / or induce killing of cancer cells expressing wild-type NPM1. In some aspects, the immune effector cell does not substantially target cancer cells expressing wild-type NPM1. In some aspects, the immune effector cell does not substantially induce killing of cancer cells expressing wild-type NPM1. In some aspects, the cell is a T cell. In some embodiments, the T cells are human CD8 + In some embodiments, the cells are T cells. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are macrophages. In some embodiments, the cancer cells are acute myeloid leukemia (AML) cells.

[0048] In some aspects, the present disclosure provides a pharmaceutical composition comprising a cell transformed with an expression vector comprising an isolated nucleic acid encoding a CAR polypeptide described herein and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a pharmaceutical composition comprising a cell expressing a CAR polypeptide described herein and a pharmaceutically acceptable carrier.

[0049] In some aspects, the present disclosure provides methods for making cells that express a CAR polypeptide described herein, the method comprising: (i) purifying the cells from peripheral blood mononuclear cells (PMBCs) of a subject; (ii) optionally activating the cells with an anti-CD3 antibody, or an antigen-binding fragment thereof, and / or an anti-CD28 antibody, or an antigen-binding fragment thereof; (iii) transducing the cells with an expression vector comprising an isolated nucleic acid encoding a CAR polypeptide described herein; (iv) isolating the cells that express the CAR polypeptide; and (v) optionally expanding the isolated cells.

[0050] In some aspects, the present disclosure provides methods for producing cells that express a CAR polypeptide described herein, the method comprising: (i) inducing pluripotent stem cells (iPSCs) to differentiate into immune effector cells; (ii) transducing the immune effector cells with an expression vector comprising an isolated nucleic acid encoding a CAR polypeptide described herein; (iii) isolating the immune effector cells that express the CAR polypeptide; and (iv) optionally, expanding the isolated immune effector cells.

[0051] In any of the above or related embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells are macrophages. In some embodiments, the immune effector cells are T cells.

[0052] In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, wherein the cell surface of cells comprising the cancer presents an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein, and the method comprises administering to the subject an antibody, or antigen-binding fragment thereof, a cell, or a pharmaceutical composition described herein, in an amount sufficient to treat the cancer. In some aspects, the cancer is acute myeloid leukemia (AML). In some aspects, the method of treating cancer is a method of reducing the amount of cancer or a method of prolonging survival in a subject.

[0053] In some aspects, the present disclosure provides a method of treating acute myeloid leukemia (AML) in a subject in need thereof, comprising administering to the subject an antibody, or antigen-binding fragment thereof, a cell, or a pharmaceutical composition described herein, in an amount sufficient to treat the AML. In some aspects, the AML is relapsed or refractory AML.

[0054] In some aspects, the present disclosure provides a method of preventing recurrence of AML in a subject in remission from AML, the method comprising administering to the subject an antibody, or antigen-binding fragment thereof, a cell, or a pharmaceutical composition described herein.

[0055] In any of the above or related aspects, the present disclosure provides a method comprising, prior to the administering step, detecting whether the subject expresses NPM1c or whether the subject has an NPM1c mutation in the NPM1 gene, and proceeding with the administering step if the subject expresses NPM1c or has an NPM1c mutation.

[0056] In any of the above or related aspects, the disclosure provides methods wherein administration is intravenous, intrathecal, intraosseous, or intraspinal.

[0057] In any of the above or related aspects, the method further comprises administering one or more additional therapeutic agents or procedures. hi some aspects, the additional therapeutic agent is an inhibitor of an immune checkpoint molecule, optionally the immune checkpoint molecule is TIM-3, PD-1, PD-L1, or CTLA-4, and optionally the inhibitor is an antibody.

[0058] In some aspects, the present disclosure provides use of an antibody, or antigen-binding fragment thereof, described herein, a CAR polypeptide described herein, a cell described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for treating cancer in a subject, wherein the cell surface of the cancer-containing cells presents an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein; optionally, the use in combination with one or more additional therapeutic agents or procedures.

[0059] In any of the above or related embodiments, the subject is a human.

[0060] In some aspects, the present disclosure provides kits that include one or more containers containing: (i) an antibody, or antigen-binding fragment thereof, described herein, a CAR polypeptide described herein, a cell described herein, or a pharmaceutical composition described herein; (ii) optionally, one or more additional therapeutic agents, and (iii) instructions for use in treating cancer in a subject.

[0061] In another aspect, described herein is an antibody or antigen-binding fragment thereof that specifically binds to an antigen comprising a neoepitope complexed with (or presented by) an MHC (e.g., MHC class I) protein (e.g., HLA-A2). In certain embodiments of this aspect, an antibody or antigen-binding fragment thereof that specifically binds to an antigen comprising a neoepitope complexed with (or presented by) an MHC (e.g., MHC class I) protein (e.g., HLA-A2) can be prepared, for example, by (i) isolating antibody clones that bind to the antigen using a yeast surface-displayed antibody (e.g., scFv) library or a phage-displayed antibody (e.g., scFv) library, and (ii) screening the antibody clones multiple times (two, three, four, or more times) using a yeast surface-displayed antibody (e.g., scFv) library or a phage-displayed antibody (e.g., scFv) library. and multiple (two, three, four, or more) rounds of negative selection (e.g., selecting for yeast or phage clones that bind to the MHC protein alone and / or in complex with a control peptide (i.e., a peptide different from the neoepitope) to select for antibody clones that specifically bind to the antigen); the selected antibody clones bind to the antigen and do not or do not substantially bind to the MHC protein alone and / or the MHC protein in complex with the control peptide. In certain embodiments of this aspect, the antigen is a dimeric neoepitope-MHC complex. In certain embodiments of this aspect, the neoepitope-MHC complex is NPM1c:HLA-A2. [Brief explanation of the drawings]

[0062] [Figure 1-1]Figures 1A-1D show the isolation of human scFv specific for the AIQ-HLA-A2 complex by yeast surface display. Figure 1A shows a schematic of the epitope peptide-HLA-A2 complex, the scFv displayed on the yeast surface, and the binding of the peptide-HLA-A2 complex to the scFv on the yeast cell surface. The schematic is adapted from Chao et al., NAT PROTOC 1: 755 (2006). [Figure 1-2] Figures 1A-1D show the isolation of human scFv specific for the AIQ-HLA-A2 complex by yeast surface display. Figures 1B-1 and 1B-2 show the strategy and steps used to isolate yeast cells displaying scFvs that specifically recognize the AIQ-HLA-A2 complex. Selection rounds are indicated on the left (rounds 1-5 in Figure 1B-1 and rounds 6-9 in Figure 1B-2). "Antigen" indicates the peptide-HLA-A2 complex or HLA-A2 alone used for positive or negative selection. In the first two rounds of selection, yeast cells were selected by magnetic cell sorting (MACS). In the remaining rounds of selection, yeast cells were sorted by flow cytometry based on staining with FITC-labeled anti-c-Myc antibody and PE-labeled anti-mouse IgG or APC-labeled streptavidin. Gates for sorted cells are shown. FACS plots are labeled #1 to #7. [Figure 1-3]Figures 1A-1D show the isolation of human scFv specific for the AIQ-HLA-A2 complex by yeast surface display. Figures 1B-1 and 1B-2 show the strategy and steps used to isolate yeast cells displaying scFvs that specifically recognize the AIQ-HLA-A2 complex. Selection rounds are indicated on the left (rounds 1-5 in Figure 1B-1 and rounds 6-9 in Figure 1B-2). "Antigen" indicates the peptide-HLA-A2 complex or HLA-A2 alone used for positive or negative selection. In the first two rounds of selection, yeast cells were selected by magnetic cell sorting (MACS). In the remaining rounds of selection, yeast cells were sorted by flow cytometry based on staining with FITC-labeled anti-c-Myc antibody and PE-labeled anti-mouse IgG or APC-labeled streptavidin. Gates for sorted cells are shown. FACS plots are labeled #1 to #7. [Figure 1-4] Figures 1A-1D show the isolation of human scFv specific for the AIQ-HLA-A2 complex by yeast surface display. Figures 1C-1 and 1C-2 show flow cytometry (FACS) data for viable cells. Sorted yeast cells from rounds 4 through 9 were expanded and then stained with FITC-labeled anti-c-Myc antibody and biotin-labeled HLA-A2, GIL-HLA-A2, SLL-HLA-A2, or AIQ-HLA-A2, followed by APC-labeled streptavidin. Viable cells (DAPI-negative) were gated by flow cytometry. Data for rounds 4 and 5 are shown in Figure 1C-1, and data for rounds 6 through 9 are shown in Figure 1C-2. FACS plots are labeled #1 through #29. [Figure 1-5]Figures 1A-1D show the isolation of human scFv specific for the AIQ-HLA-A2 complex by yeast surface display. Figures 1C-1 and 1C-2 show flow cytometry (FACS) data for viable cells. Sorted yeast cells from rounds 4 through 9 were expanded and then stained with FITC-labeled anti-c-Myc antibody and biotin-labeled HLA-A2, GIL-HLA-A2, SLL-HLA-A2, or AIQ-HLA-A2, followed by APC-labeled streptavidin. Viable cells (DAPI-negative) were gated by flow cytometry. Data for rounds 4 and 5 are shown in Figure 1C-1, and data for rounds 6 through 9 are shown in Figure 1C-2. FACS plots are labeled #1 through #29. [Figure 1-6] Figures 1A-1D show the isolation of human scFv specific for the AIQ-HLA-A2 complex by yeast surface display. Figure 1D shows flow cytometry data. Yeast cells expressing the YG1 or YG2 clone were stained and analyzed as in Figure 1C. The percentages in Figures 1B, 1C, and 1D indicate the percentage of cells within the gated region. [Figure 2-1] Figures 2A-2E show the specific and high-affinity binding of YG1 scFv to the AIQ-HLA-A2 complex on AML cells. Figure 2A shows a schematic diagram of a switchable yeast display / secretion vector for expressing scFv-Fc fusion proteins. In this switchable system, scFv-Fc can be secreted or displayed on yeast cells depending on whether OmeY is added to the culture (Van Deventer et al., PROTEIN ENG DES SEL 28: 317 (2015)). Figure 2B shows SDS-PAGE analysis of purified YG1 scFv-Fc protein. Lane 1: protein ladder, Lane 2: non-reduced scFv-Fc protein (1 μg), Lane 3: reduced scFv-Fc protein (1 μg). The gel was stained using Coomassie blue. [Figure 2-2]Figures 2A-2E show the specific and high-affinity binding of YG1 scFv to the AIQ-HLA-A2 complex on AML cells. Figure 2C shows flow cytometry data of HLA-A2 expression by OCI-AML3, T2, GMB, PC-3, and OCI-AML2 cells. Dark-shaded histograms show staining with anti-HLA-A2, and light-shaded histograms show staining with an isotype control antibody. Figure 2D shows flow cytometry data of AIQ-HLA-A2 expression by OCI-AML3, T2, GMB, and PC-3 cells. Dark-shaded histograms show staining with YG1 scFv-Fc and anti-HA, and light-shaded histograms show staining with BSA followed by anti-HA. Representative data from triplicate measurements are shown. [Figure 2-3] Figures 2A-2E show the specific and high-affinity binding of YG1 scFv to the AIQ-HLA-A2 complex on AML cells. Figure 2E shows the kinetic analysis of the interaction between YG1 scFv-Fc and AIQ-HLA-A2, SLL-HLA-A2, or HLA-A2 by biolayer interferometry. A streptavidin biosensor chip on a ForteBio Octet RED 96 was coated with biotinylated YG1 scFv-Fc protein. The binding to scFv-Fc (association) was measured by immersing the chip in increasing concentrations (shown below the binding curves) of AIQ-HLA-A2, SLL-HLA-A2, or HLA-A2, followed by transfer to wells containing buffer to measure the dissociation rate (dissociation). Representative data from three separate experiments are shown. [Figure 3-1] Figures 3A-3D show the generation of NPM1c-CAR-T cells (containing YG1 scFv) specific for the AIQ-HLA-A2 complex. Figure 3A shows a schematic of the CAR vector consisting of the scFv (YG1 or CD19), CD8α extracellular hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3ζ activation domain, followed by self-cleaving P2A and EGFP. Figure 3B shows a schematic of the recognition of the AIQ-HLA-A2 complex on AML cells by NPM1c CAR-T cells. [Figure 3-2]Figures 3A-3D show the generation of NPM1c-CAR-T cells (containing YG1 scFv) specific for the AIQ-HLA-A2 complex. Figure 3C shows flow cytometry data of CAR expression by untransduced and transduced T cells. Transduced T cells were enriched and expanded by sorting for GFP+ cells and stained with AF647-labeled anti-human IgG heavy and light chain antibodies that recognize the scFv. Untransduced T cells were activated and expanded without sorting. The GFP and anti-human IgG staining profiles of live cells (DAPI-) are shown. Figure 3D shows flow cytometry data demonstrating that NPM1c CAR-T cells recognize the AIQ-HLA-A2 complex. Untransduced and transduced T cells were incubated with biotinylated AIQ-HLA-A2, SLL-HLA-A2, or HLA-A2 complexes, followed by staining with streptavidin-APC. GFP and streptavidin-APC staining profiles of viable (DAPI-) untransduced T cells, NPM1c CAR-T cells, and CD19 CAR-T cells are shown. Data in Figures 3C and 3D are representative of at least three independent experiments. Percentages indicate the percentage of cells in the gated region. [Figure 4-1]Figures 4A-4J show that NPM1c CAR-T cells (containing YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figures 4A-4B show that NPM1c CAR-T cells kill target cells in vitro. NPM1c CAR-T cells were cocultured with OCI-AML3, GMB, and PC-3 tumor cells at the indicated effector:target (E:T) ratios for 24 hours. The cell mixtures were stained for CD8 and CD33, CD19, or mCherry, followed by flow cytometry. The percentage of CAR-T cells was quantified by CD8 staining, the percentage of OCI-AML3 cells by CD33, the percentage of GMB cells by CD19, and the percentage of PC-3 cells by mCherry. The percentage of specific tumor cell lysis was calculated (see Materials and Methods in the Examples for formula). The staining profiles of CD8 versus CD33, CD19, or mCherry at different E:T ratios (Figure 4A) and the percentage of specific lysis (Figure 4B) are shown. The percentages of cells in the gated regions are shown. p values ​​represent a comparison between NPM1c CAR-T cells and non-transduced T cells at the same E:T ratio. [Figure 4-2] Figure 4A (continued). [Figure 4-3] Figure 4A (continued). [Figure 4-4] Figure 4A (continued). [Figure 4-5]Figures 4A-4J show that NPM1c CAR-T cells (containing YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figures 4A-4B show that NPM1c CAR-T cells kill target cells in vitro. NPM1c CAR-T cells were cocultured with OCI-AML3, GMB, and PC-3 tumor cells at the indicated effector:target (E:T) ratios for 24 hours. The cell mixtures were stained for CD8 and CD33, CD19, or mCherry, followed by flow cytometry. The percentage of CAR-T cells was quantified by CD8 staining, the percentage of OCI-AML3 cells by CD33, the percentage of GMB cells by CD19, and the percentage of PC-3 cells by mCherry. The percentage of specific tumor cell lysis was calculated (see Materials and Methods in the Examples for formula). The staining profiles of CD8 versus CD33, CD19, or mCherry at different E:T ratios (Figure 4A) and the percentage of specific lysis (Figure 4B) are shown. The percentages of cells in the gated regions are shown. p values ​​represent a comparison between NPM1c CAR-T cells and non-transduced T cells at the same E:T ratio. [Figure 4-6] Figures 4A-4J show that NPM1c CAR-T cells (containing YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figure 4C shows a comparison of IFN-γ and IL-2 expression between CAR-T cells and non-transduced T cells. NPM1c CAR-T cells and non-transduced T cells were co-cultured with OCI-AML3, GMB, or PC-3 in the presence of monensin and brefeldin A for 12 hours. Cells were stained for CD3, then permeabilized and stained for intracellular IFN-γ or IL-2, followed by flow cytometry. The percentages of IFN-γ+ or IL-2+ NPM1c CAR-T cells and non-transduced T cells are shown. P values ​​are indicated. [Figure 4-7]Figures 4A-4J show that NPM1c CAR-T cells (containing YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figure 4D shows that NPM1c CAR-T cells secrete multiple cytokines upon stimulation by NPM1c+HLA-A2+ target cells. NPM1c CAR-T cells or untransduced T cells were co-cultured with NPM1c+HLA-A2+OCI-AML3 cells for 16 hours. Culture supernatants were collected and simultaneously assayed for 20 different cytokines using the Quantibody Human Cytokine Array. Each cytokine contained quadruplicate antibody spots. T cells from four different healthy donors were analyzed individually. n=4 replicate antibody spots. Graph bars and error bars represent the mean ± SE. p values ​​are indicated. [Figure 4-8] Figure 4D (continued). [Figure 4-9] Figures 4A-4J show that NPM1c CAR-T cells (containing YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figures 4E-4F show that NPM1c CAR-T cells proliferate in response to NPM1c+HLA-A2+ target cells. NPM1c CAR-T cells or untransduced T cells were cocultured with OCI-AML3 cells for 5 days. The absolute cell numbers of CAR-T cells or untransduced T cells were determined by flow cytometry using precision counting beads. Ki-67 expression by NPM1c CAR-T cells or untransduced T cells was assayed by intracellular staining followed by flow cytometry. Figure 4E provides a comparison of the numbers of NPM1c CAR-T cells and untransduced T cells at day 5. Figure 4F provides a comparison of the mean fluorescence intensity (MFI) of intracellular Ki-67 staining between non-transduced T cells and NPM1c CAR-T cells. p-values ​​indicate the comparison between NPM1c CAR-T cells and non-transduced T cells. n=5 biologically independent samples. Graph bars and error bars represent the mean ± SE. [Figure 4-10]Figures 4A-4J show that NPM1c CAR-T cells (containing YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figures 4G-4H show that NPM1c CAR-T cells specifically kill HLA-A2+NPM1c+ human tumor cells in vitro. OCI-AML2 (HLA-A2 positive) and PC-3 (HLA-A2 negative) cells were transduced with lentivirus expressing NPM1c (lenti-NPM1c) or an empty negative control (lenti-NC). Transduced cells were sorted and expanded. Specific killing of transduced or control OCI-AML2 cells (Figure 4G) and PC-3 cells (Figure 4H) by NPM1c CAR-T cells or untransduced T cells is compared. NPM1c CAR-T cells or non-transduced T cells were co-cultured with transduced or control OCI-AML2 or PC-3 target cells at the indicated E:T ratios for 24 hours. Target cell killing was measured by assaying luciferase activity of surviving target cells. The percentage of specific lysis of tumor cells at different E:T ratios was calculated. Target cells and T cells in each reaction are shown. [Figure 4-11] Figures 4A-4J show that NPM1c CAR-T cells (containing YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figure 4I shows flow cytometry analysis of YG1 scFv-FC binding to T2 cells pulsed with different concentrations of AIQ peptide (left panel) or SLL peptide (right panel). [Figure 4-12]Figures 4A-4J show that NPM1c CAR-T cells (containing YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figure 4J shows a comparison of specific killing of T2 cells pulsed with different concentrations of AIQ peptide (left panel) or SLL peptide (right panel) by NPM1c CAR-T cells or non-transduced T cells. NPM1c CAR-T cells or non-transduced T cells were cocultured with peptide-pulsed T2 target cells at the indicated E:T ratios for 24 hours. Target cell killing was measured by assaying luciferase activity of surviving target cells. p values ​​indicate a comparison between NPM1c CAR-T cells and non-transduced T cells at the same E:T ratio. n = 3 biologically independent samples. Data points and error bars represent the mean ± SE. [Figure 5-1] Figures 5A-5H show that NPM1c CAR-T (containing YG1 scFv) therapy reduces leukemia burden and prolongs survival in mice bearing NPM1c-positive AML cells, but not in mice bearing NPM1c-negative AML cells. Figure 5A shows the experimental design. NSG mice were intravenously injected with OCI-AML3 cells (1 × 10) or GMB cells (2 × 10) (D-4) and imaged for engraftment 4 days later (D0). Mice were then intravenously injected with 1 × 10 NPM1c CAR-T cells, untransduced T cells, or CD19 CAR-T cells. Mice were monitored every 3 days by bioluminescence imaging to assess tumor burden and survival. [Figure 5-2]Figures 5A–5H show that NPM1c CAR-T (containing YG1 scFv) therapy reduces leukemia burden and prolongs survival in mice bearing NPM1c-positive AML cells, but not in mice bearing NPM1c-negative AML cells. Figure 5B shows a comparison of OCI-AML3 leukemia burden measured by bioluminescence imaging between mice treated with NPM1c CAR-T cells and mice treated with untransduced T cells at the indicated days (D0–D18) after T cell injection (n=5). Comparison of total flux (luciferase signal from systemic OCI-AML3 leukemia cells) for each experimental group is shown. Experiments were repeated twice with four or five mice per group. Figure 5C shows Kaplan-Meier survival curves (n=9) for mice treated with NPM1c CAR-T cells or untransduced T cells as in Figure 5B. P values ​​are indicated. [Figure 5-3]Figures 5A-5H show that NPM1c CAR-T (containing YG1 scFv) therapy reduces leukemia burden and prolongs survival in mice with NPM1c-positive AML cells, but not in mice with NPM1c-negative AML cells. Figure 5D shows a comparison of GMB lymphoma burden measured by bioluminescence imaging between mice treated with NPM1c CAR-T cells, non-transduced T cells, and CD19 CAR-T cells on the indicated days (D0-D21) after T cell injection (n = 3-5). Comparison of total flux (luciferase signal from systemic GMB cells) for each experimental group is shown. Experiments were repeated twice with 3-5 mice per group. P values ​​for total flux were p = 0.992 for non-transduced T cells and NPM1c CAR-T, p = 0.003 for CD19 CAR-T and non-transduced T cells, and p = 0.047 for CD19 CAR-T and NPM1c CAR-T. Figure 5E shows Kaplan-Meier survival curves (n = 3–5) for mice treated with NPM1c CAR-T cells, non-transduced T cells, or CD19 CAR-T cells as in Figure 5D. P values: p = 0.124 for non-transduced T cells and NPM1c CAR-T, p = 0.012 for CD19 CAR-T and non-transduced T, and p = 0.015 for CD19 CAR-T and NPM1c CAR-T. [Figure 5-4]Figures 5A-5H show that NPM1c CAR-T (containing YG1 scFv) therapy reduces leukemia burden and prolongs survival in mice with NPM1c-positive AML cells, but not in mice with NPM1c-negative AML cells. Figure 5F provides a comparison of OCI-AML3 leukemia burden measured by bioluminescence imaging on the indicated days (D0-D21) after T cell / PBS injection in mice administered NPM1c CAR-T cells, untransduced T cells, or PBS (n = 3-4). Comparison of total flux (luciferase signal from OCI-AML3 cells throughout the body) is shown. P values ​​are p = 0.395 for PBS vs. untransduced T, p = 0.018 for PBS vs. NPM1c CAR-T, and p = 0.011 for untransduced T vs. NPM1c CAR-T. Figure 5G provides a comparison of OCI-AML2 leukemia burden measured by bioluminescence imaging on the indicated days (D0–D21) after T cell injection between mice treated with NPM1c CAR-T cells (n = 5) and mice treated with non-transduced T cells (n = 5). A comparison of total flux (luciferase signal from OCI-AML2 cells throughout the body) is shown. Figure 5H provides Kaplan-Meier survival curves for the mice shown in Figure 5G treated with NPM1c CAR-T cells (n = 5) or non-transduced T cells (n = 5). Data points and error bars represent the mean ± SE. p values ​​are indicated. [Figure 6-1] Figures 6A-6I show that NPM1c CAR-T cells (containing YG1 scFv) reduce leukemia burden in the blood, spleen, bone marrow, and liver. Figure 6A shows a comparison of OCI-AML3 leukemia burden measured by bioluminescence imaging between NSG mice injected with OCI-AML3 AML cells and then untransduced T cells, or NSG mice injected with OCI-AML3 AML cells and then NPM1c CAR-T cells. A comparison of total flux (luciferase signal from OCI-AML3 cells throughout the body) is shown. Mice (n = 5) were imaged on the day of T cell injection (day 0) and 18 days later. [Figure 6-2]Figures 6A-6I show that NPM1c CAR-T cells (containing YG1 scFv) reduce leukemia burden in the blood, spleen, bone marrow, and liver. Figures 6B-6C show representative flow cytometry plots illustrating the gating strategy and expression profiles for cell populations obtained from the mouse depicted in Figure 6A. Blood, spleen, bone marrow, and liver were collected on day 18 to prepare single-cell suspensions, which were stained for mouse CD45 and human CD45, CD8, CD33, PD-1, and Tim-3, followed by flow cytometry. Representative staining profiles and gating strategies for blood and spleen (Figure 6B) and bone marrow and liver (Figure 6C) are shown. The gating strategy included gating mCD45 vs. hCD45 in live cells (DAPI-), gating hCD33 vs. hCD8 in hCD45+ cells, gating hPD-1 vs. hCD8 in hCD8+ cells, and gating hTim-3 vs. hCD8 in hCD8+ cells. Numbers indicate the percentage of cells within the gated area. [Figure 6-3] Figures 6A-6I show that NPM1c CAR-T cells (containing YG1 scFv) reduce leukemia burden in the blood, spleen, bone marrow, and liver. Figures 6B-6C show representative flow cytometry plots illustrating the gating strategy and expression profiles for cell populations obtained from the mouse depicted in Figure 6A. Blood, spleen, bone marrow, and liver were collected on day 18 to prepare single-cell suspensions, which were stained for mouse CD45 and human CD45, CD8, CD33, PD-1, and Tim-3, followed by flow cytometry. Representative staining profiles and gating strategies for blood and spleen (Figure 6B) and bone marrow and liver (Figure 6C) are shown. The gating strategy included gating mCD45 vs. hCD45 in live cells (DAPI-), gating hCD33 vs. hCD8 in hCD45+ cells, gating hPD-1 vs. hCD8 in hCD8+ cells, and gating hTim-3 vs. hCD8 in hCD8+ cells. Numbers indicate the percentage of cells within the gated area. [Figure 6-4] Figures 6A-6I show that NPM1c CAR-T cells (containing YG1 scFv) reduce leukemia burden in the blood, spleen, bone marrow, and liver. Figure 6D shows a comparison of the total numbers of hCD33+ leukemia cells and hCD8+ T cells in different tissues between mice administered NPM1c CAR-T cells and non-transduced T cells (left bars represent treatment with non-transduced T cells, right bars represent treatment with NPM1c CAR-T cells). [Figure 6-5] Figures 6A-6I show that NPM1c CAR-T cells (containing YG1 scFv) reduce leukemia burden in the blood, spleen, bone marrow, and liver. Figure 6E shows a comparison of the percentage of hCD33+ leukemia cells and hCD8+ T cells among hCD45+ cells in different tissues between mice administered NPM1c CAR-T cells and mice administered non-transduced T cells (left bars represent treatment with non-transduced T cells, right bars represent treatment with NPM1c CAR-T cells). [Figure 6-6] Figures 6A-6I show that NPM1c CAR-T cells (containing YG1 scFv) reduced leukemia burden in the blood, spleen, bone marrow, and liver. Figure 6F shows the ratio of the percentage of hCD8+ T cells to hCD33+ leukemia cells in different tissues of mice administered NPM1c CAR-T cells or non-transduced T cells (the left bar represents treatment with non-transduced T cells, and the right bar represents treatment with NPM1c CAR-T cells). Figures 6G-6H show the percentage of PD1+ T cells (Figure 6G) or Tim-3+ T cells (Figure 6H) among human CD8+ T cells in different tissues (the left bar represents treatment with non-transduced T cells, and the right bar represents treatment with NPM1c CAR-T cells). P values ​​are shown in Figures 6A, 6D, 6E, 6F, 6G, and 6H (n=5). [Figure 6-7]Figures 6A-6I show that NPM1c CAR-T cells (containing YG1 scFv) reduce leukemia burden in the blood, spleen, bone marrow, and liver. Figure 6I shows that NPM1c CAR T cells effectively eliminate leukemia cells in the bone marrow 30 days after CAR-T cell injection. NSG mice were transplanted with OCI-AML3 and injected with NPM1c CAR-T cells, untransduced T cells, or PBS 4 days later (mice are shown in Figure 5F). Cells were collected from the bone marrow of surviving mice 30 days after T cell injection. Cells were stained for mouse CD45 and human CD45, CD8, and CD33, followed by flow cytometry. Shown are FSC and DAPI staining profiles for total cells (left), hCD45 and mCD45 staining profiles gated on live (DAPI-) cells (middle), and hCD33 and hCD8 staining profiles for hCD45+ cells (right). Numbers indicate the percentage of cells within the gated region. [Figure 6-8] Figure 6I (continued). [Figure 6-9] Figure 6I (continued). [Figure 6-10] Figure 6I (continued). [Figure 7-1] Figures 7A-7G show that NPM1c CAR-T cells (containing YG1 scFv) effectively kill primary human AML blasts in vitro and in vivo but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7A shows that NPM1c CAR-T cells kill NPM1c+HLA-A2+ primary AML blasts from three donors in vitro. NPM1c CAR-T cells or untransduced T cells were incubated with AML blasts at the indicated ratios for 24 hours. The absolute number of AML blasts was quantified by staining for CD8 and CD33 followed by flow cytometry using Precision Count beads. The percentage of specific lysis of tumor cells at different E:T ratios was calculated. n=3 biological replicates. Graph bars and error bars represent the mean ± SE. p values ​​are indicated. [Figure 7-2] Figures 7A-7G show that NMP1c CAR-T cells (containing YG1 scFv) effectively kill primary human AML blasts in vitro and in vivo but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7B shows flow cytometry analysis of HLA-A2 expression by HSPCs. Human CD34+ HSPCs were purified from two donor fetal livers using the EasySep Human CD34 Positive Selection Kit. Dark histograms are stained with anti-HLA-A2, and light histograms are stained with an isotype control antibody. Representative data from technical triplicates are shown. Figure 7C shows flow cytometry analysis of HSPCs for YG1 scFv-Fc binding. Dark-shaded histograms are stained with YG1 scFv-Fc and anti-HA, and light-shaded histograms are stained with BSA followed by anti-HA. Representative data from three separate experiments with technical triplicates are shown. [Figure 7-3] Figures 7A-7G show that NPM1c CAR-T cells (containing YG1 scFv) effectively kill primary human AML blast cells in vitro and in vivo but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7D shows that NPM1c CAR-T cells do not kill HLA-A2+CD34+ HSPCs. NPM1c CAR-T cells and untransduced T cells were incubated with HSPCs at the indicated E:T ratios for 24 hours. The cell mixtures were stained for CD8 and CD34 and quantified by flow cytometry using Precision Count beads. Examples of CD8 (T cells) and CD34 (HSPC) staining profiles at different E:T ratios are shown. The percentages of cells within the gated regions are indicated. [Figure 7-4]Figures 7A-7G show that NMP1c CAR-T cells (containing YG1 scFv) effectively kill primary human AML blast cells in vitro and in vivo, but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7E shows a comparison of specific cell lysis between NPM1c CAR-T cells and non-transduced T cells at different E:T ratios. p values ​​indicate a comparison between NPM1c CAR-T cells and non-transduced T cells at the same E:T ratio. n=3 biological replicates. Data points and error bars represent the mean ± SE. [Figure 7-5] Figures 7A-7G show that NPM1c CAR-T cells (containing YG1 scFv) effectively kill primary human AML blasts in vitro and in vivo but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7F shows that treatment with NPM1c CAR-T cells reduces leukemic burden in primary HLA-A2+NPM1c+ AML xenografts. NSGS mice were transplanted with human AML blasts. Two weeks later, when AML blasts were detectable in the blood, the mice were administered NPM1c CAR-T cells or untransduced T cells. Blood was collected from the mice on the indicated days after T cell transfer, and mononuclear cells were stained for mCD45, hCD45, and hCD8. Representative staining profiles for hCD45 and mCD45, gated on live hCD8- cells, are shown. AML blasts were hCD45+hCD8-. Numbers indicate the percentage of cells within the gated region. [Figure 7-6]Figures 7A-7G show that NPM1c CAR-T cells (containing YG1 scFv) effectively kill primary human AML blasts in vitro and in vivo but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7G shows a comparison of the percentage of hCD45+CD8- AML blasts in peripheral blood between mice administered NPM1c CAR-T cells and mice administered untransduced T cells. AML blast levels were measured before T cell injection (day 0) and on days 9 and 18 after T cell injection. The left bar represents treatment with untransduced T cells, and the right bar represents treatment with NPM1c CAR-T cells. Graph bars and error bars represent the mean ± SE. p values ​​(two-tailed independent-samples t-test) are shown (n = 5). DETAILED DESCRIPTION OF THE INVENTION

[0063] The present disclosure is based, at least in part, on the identification of single-chain variable antibody fragments (scFvs) that specifically and with high affinity bind to NPM1c neoepitopes complexed with HLA-A2. The present disclosure provides novel scFvs, antibodies, and antigen-binding fragments thereof, that bind to such NPM1c neoepitopes complexed with HLA-A2. In addition, the present disclosure provides bispecific binding molecules based on the scFvs of the present disclosure that specifically bind to such NPM1c neoepitopes complexed with HLA-A2 and further bind to another target.

[0064] Additionally, the present disclosure provides a chimeric antigen receptor (CAR) polypeptide comprising an extracellular binding domain that specifically binds to an antigen comprising an NPM1c neoepitope complexed with HLA-A2.

[0065] Additionally, the present disclosure provides T cells expressing a CAR polypeptide comprising an extracellular binding domain that specifically binds to an antigen comprising an NPM1c neoepitope in complex with HLA-A2. As described herein and shown in the Examples presented herein, T cells expressing a CAR polypeptide comprising an extracellular binding domain that specifically binds to an antigen comprising an NPM1c neoepitope in complex with HLA-A2 specifically kill AML cells in vitro and reduce leukemia burden and prolong survival in vivo in AML mouse models.

[0066] Therefore, antibodies and antigen-binding fragments thereof, bispecific molecules, CAR polypeptides, and T cells expressing the CAR polypeptides described herein are useful for targeted immunotherapy to treat cancers carrying NPM1c mutations. For example, the antibodies and antigen-binding fragments thereof, CAR polypeptides, and T cells expressing the CAR polypeptides disclosed herein are useful for targeted immunotherapy to treat acute myeloid leukemia (AML). In one aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an antigen comprising an NPM1c neoepitope when such epitope is complexed with (or presented by) a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2). In one aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to one or more neoepitopes having the following amino acid sequences: AIQDLCLAV (SEQ ID NO: 1), AIQDLCVAV (SEQ ID NO: 71), CLAVEEVSL (SEQ ID NO: 72), VEEVSLRK (SEQ ID NO: 73), AVEEVSLR (SEQ ID NO: 74), AVEEVSLRK (SEQ ID NO: 75), CLAVEEVSLRK (SEQ ID NO: 76) when such epitopes are complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2). In one aspect, provided herein is an antibody or antigen-binding fragment thereof that does not bind, or does not substantially bind, to an MHC class I protein alone. In one aspect, provided herein is an antibody or antigen-binding fragment thereof that does not bind or does not substantially bind to a control peptide complexed with an MHC class I protein (e.g., the control peptide is an NY-ESO-1 epitope (e.g., a peptide comprising SEQ ID NO: 62) or an influenza virus M1 epitope (e.g., a peptide comprising SEQ ID NO: 63)). In one aspect, provided herein is an antibody or antigen-binding fragment thereof that does not bind or does not substantially bind to an NPM1c neoepitope alone (without an MHC class I protein).In some embodiments, the NPM1c neoepitope comprises the amino acid sequence AIQDLCLAV (SEQ ID NO: 1), and the MHC class I protein is an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele). In some embodiments, the antigen is on the surface of a cancer cell (e.g., when the cancer is NPM1c+, e.g., when the cancer is AML). In one embodiment, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an amino acid sequence comprising AIQDLCLAV (SEQ ID NO: 1) complexed with an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele). The antibodies and antigen-binding fragments thereof provided herein are described below.

[0067] In one aspect, provided herein is a bispecific molecule comprising: (i) a first binding domain that specifically binds to an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2), and (ii) a second antigen-binding domain that specifically binds to a second antigen. In some aspects, the second antigen is an antigen expressed on T cells or natural killer cells. In some aspects, the second antigen is CD3 (e.g., human CD3), NKp46 (e.g., human NKp46), or CD16A (e.g., human CD16A). In one aspect, provided herein is a bispecific molecule comprising: (i) a first antigen-binding domain that specifically binds to an amino acid sequence comprising AIQDLCLAV (SEQ ID NO: 1) complexed with an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele), and (ii) a second antigen-binding domain that specifically binds to a second antigen. The bispecific molecules provided herein are described below.

[0068] In one aspect, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein (and optionally a pharmaceutically acceptable carrier). In one aspect, provided herein is a pharmaceutical composition comprising a bispecific molecule described herein (and optionally a pharmaceutically acceptable carrier). The pharmaceutical compositions provided herein are described below.

[0069] In one aspect, provided herein is a chimeric antigen receptor (CAR) polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain specifically binds to an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2). In one aspect, provided herein is a CAR polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain specifically binds to an amino acid sequence comprising AIQDLCLAV (SEQ ID NO: 1) complexed with an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele). In one aspect, provided herein is a CAR polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain comprises any antibody or antigen-binding fragment thereof described herein. In one aspect, provided herein is a CAR polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain comprises any bispecific molecule described herein. The CAR polypeptides provided herein are described below.

[0070] In one aspect, provided herein is an immune effector cell expressing a CAR polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain specifically binds to an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2). In one aspect, provided herein is an immune effector cell expressing a CAR polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain specifically binds to an amino acid sequence comprising AIQDLCLAV (SEQ ID NO: 1) complexed with an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele). In one aspect, provided herein is an immune effector cell expressing a CAR polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain comprises any antibody or antigen-binding fragment thereof described herein. In one aspect, provided herein is an immune effector cell that expresses a CAR polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain comprises any of the bispecific molecules described herein. In one aspect, provided herein is an immune effector cell that expresses a CAR polypeptide described herein. In one aspect, the immune effector cell is a T cell (e.g., CD8 +T cells), natural killer cells, or macrophages. In one embodiment, expression of the CAR polypeptide causes immune effector cells to target cancer cells (e.g., the cancer is AML) that present on their surface an NPM1c neoepitope (e.g., HLA-A2) complexed with a class I major histocompatibility complex (MHC class I) protein. In one embodiment, expression of the CAR polypeptide causes immune effector cells to target cancer cells (e.g., the cancer is AML) that present on their surface the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) complexed with an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele). Immune effector cells provided herein are described below.

[0071] In one aspect, provided herein is a pharmaceutical composition comprising any of the immune effector cells described herein (and optionally a pharmaceutically acceptable carrier).

[0072] In one aspect, provided herein is a method of treating cancer in a subject (e.g., a human), the cell surface of which cells comprising the cancer presents an NPM1c neoepitope in complex with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2), and the method comprises administering to the subject any of the antibodies or antigen-binding fragments described herein. In one aspect, provided herein is a method of treating cancer in a subject (e.g., a human), the cell surface of which cells comprising the cancer presents an NPM1c neoepitope in complex with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2), and the method comprises administering to the subject any of the immune effector cells comprising a CAR polypeptide described herein. In one aspect, provided herein is a method of treating cancer in a subject (e.g., a human), the method comprising administering to the subject any antibody or antigen-binding fragment described herein, wherein the cell surface of a cell comprising the cancer displays an amino acid sequence comprising AIQDLCLAV (SEQ ID NO: 1) complexed with an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele). In one aspect, provided herein is a method of treating cancer in a subject (e.g., a human), the method comprising administering to the subject any immune effector cell wherein the cell surface of a cell comprising the cancer displays an amino acid sequence comprising AIQDLCLAV (SEQ ID NO: 1) complexed with an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele), and comprising a CAR polypeptide described herein.

[0073] In some aspects, provided herein are methods of treating NPM1c-positive cancer in a subject (e.g., a human), comprising administering to the subject any of the antibodies or antigen-binding fragments described herein. In some aspects, provided herein are methods of treating NPM1c-positive cancer in a subject (e.g., a human), comprising administering to the subject any of the immune effector cells comprising a CAR polypeptide described herein.

[0074] In one aspect, provided herein is a method of treating AML in a subject (e.g., a human), the method comprising administering to the subject any of the antibodies or antigen-binding fragments described herein. In one aspect, provided herein is a method of treating AML in a subject (e.g., a human), the method comprising administering to the subject any of the immune effector cells comprising a CAR polypeptide described herein.

[0075] Methods of treatment using the antibodies or antigen-binding fragments thereof provided herein, the CAR polypeptides provided herein, immune effector cells comprising the CAR polypeptides provided herein, and pharmaceutical compositions comprising the same provided herein, their uses, and kits comprising them are described below.

[0076] The present disclosure is also based, at least in part, on the identification of scFvs that specifically bind to neoepitopes (particularly cancer neoepitopes) complexed with MHC proteins (particularly MHC class I proteins, e.g., HLA-A2). The isolation of such specific, high-affinity antibodies was surprising because the development of antibodies and antigen-binding fragments thereof that specifically bind to neoepitope-MHC complexes presents numerous challenges. In particular, neoepitope peptides readily dissociate from MHC proteins, making it difficult to generate antibodies specific to these complexes. Without being bound by any theory, it has been discovered that the use of multiple rounds of positive and negative selection using yeast surface display technology for yeast populations that specifically bind to neoepitope-MHC complexes without binding to MHC proteins alone or in complex with a control peptide results in the identification of scFvs that specifically bind to neoepitope-MHC complexes. The selection steps are described in Example 1 and Appendix 1. Without being bound by any theory, the use of dimeric neoepitope-MHC complexes as antigens may also have contributed to the successful isolation of high affinity specific scFvs.

[0077] Thus, in one aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an antigen comprising a neoepitope (e.g., a cancer neoepitope) complexed with (or presented by) an MHC (e.g., MHC class I) protein (e.g., HLA-A2). In certain embodiments of this aspect, the antibody or antigen-binding fragment is generated using a yeast surface-display antibody (e.g., scFv) library or a phage-display antibody (e.g., scFv) library, and optionally enriched for specific binders to the neoepitope-MHC complex using multiple (two, three, four, or more) rounds of positive selection (selecting for a population of yeast or phage that binds to the antigen) and multiple (two, three, four, or more) rounds of negative selection (e.g., selecting for and removing a population of yeast or phage that binds to the MHC protein alone and / or in complex with a control peptide (i.e., a peptide different from the neoepitope)). In certain embodiments of this aspect, antibodies or antigen-binding fragments are generated using a yeast surface-display antibody (e.g., scFv) library or a phage-display antibody (e.g., scFv) library and, optionally, enriched for specific binders to the neoepitope-MHC complex using multiple rounds (at least three, at least four, or at least five) of positive selection (selecting for populations of yeast or phage that bind to the antigen) and multiple rounds (at least two, at least three, or at least four) of negative selection (e.g., selecting for and removing populations of yeast or phage that bind to the MHC protein alone and / or in complex with a control peptide (e.g., selecting for and removing populations of yeast or phage that bind to the MHC protein alone at least twice, and selecting for and removing populations of yeast or phage that bind to the MHC protein in complex with a control peptide at least twice)). In certain embodiments, the cell population is expanded after the selection steps (e.g., after one or more positive selection steps, after one or more negative selection steps, or after each selection step). In certain embodiments of this aspect, the antigen isDimeric neoepitope-MHC complexes (particularly those having two neoepitope:MHC molecules linked via, e.g., IgG Fc, e.g., mouse or human IgG1) are used. In certain embodiments, yeast display methods and libraries are used. In certain embodiments, positive selection involves selection (e.g., at least one or at least two rounds of selection) of a population of yeast or phage that stains for both the antigen (neoepitope-MHC complex) and the antibody (e.g., scFv). In certain embodiments, the population of yeast or phage is selected by magnetic sorting (MACS) and / or flow cytometry sorting using labeled antigen and antibody / fragment (e.g., scFv) molecules. For example, an antigen may be biotinylated by covalently attaching biotin to the antigen (which is detectable by a secondary agent conjugated to a fluorophore, e.g., streptavidin), and / or may be bound to an IgG molecule (which is detected by a labeled antibody specific for IgG), and an antibody (e.g., scFv) may be bound to an epitope tag (e.g., hemagglutinin or c-Myc) (which is detected by a fluorescently labeled antibody against the epitope tag). In one embodiment, at least three, four, five, six, seven, eight, or nine of the following selection steps are used to generate antibodies or antigen-binding fragments thereof that specifically bind to neoepitope:MHC complexes: (i) positive selection of antibody-bearing clones / cells that bind to labeled neoepitope:MHC complexes (e.g., by selecting via labeled neoepitope:MHC complexes); (ii) negative selection (selection-out) of antibody-bearing clones / cells that bind to control peptide-MHC complexes (e.g., by selecting antibody-bearing clones / cells that are not bound by labeled control peptide-MHC complexes); (iii) positive selection of antibody-bearing clones that bind to neoepitope-MHC (e.g., by selecting antibody-bearing clones / cells that double-positively stain for antibody (e.g., scFv) and neoepitope-MHC complexes); (iv) positive selection of antibody-bearing clones / cells that bind to labeled neoepitope:MHC complexes (e.g., by selecting antibody-bearing clones / cells that double-positively stain for antibody (e.g., scFv) and neoepitope-MHC complexes);(v) negative selection (selection out) of antibody-bearing clones / cells that bind to a control peptide-MHC complex or to an MHC complex alone (e.g., by selecting antibody-bearing clones / cells that are not bound by a labeled control peptide-MHC complex or a labeled MHC protein); (vi) positive selection of antibody-bearing clones that bind to neoepitope-MHC (e.g., by selecting antibody-bearing clones / cells that stain for a neoepitope-MHC complex or by double-positive staining for antibody (e.g., scFv) expression and neoepitope-MHC complex). (vii) negative selection (screening out) of antibody-bearing clones / cells that bind to the MHC complex alone (e.g., by selecting antibody-bearing clones / cells that are not bound by the labeled MHC protein); (viii) positive selection of antibody-bearing clones / cells that bind to the neoepitope-MHC complex (e.g., by selecting antibody-bearing clones / cells that double-positively stain for the antibody (e.g., scFv) and the neoepitope-MHC complex); (ix) negative selection (screening out) of antibody-bearing clones / cells that bind to the MHC complex alone (e.g., by selecting antibody-bearing clones / cells that are not bound by the labeled MHC protein). In some embodiments, at least five, six, seven, eight, or nine of the steps listed in the previous sentence are used in a specific antibody or fragment selection process, comprising at least two positive selection steps and at least two negative selection steps. In some embodiments, the at least six, seven, eight, or nine steps include at least two (preferably at least three) positive selection steps for neoepitope-MHC complexes, at least one (preferably at least two) negative selection step to select out clones / cells that bind to control peptide-MHC complexes, and at least one (preferably at least two) negative selection step to select out clones / cells that bind to the MHC protein alone.

[0023] The neoepitope is used in a selection process for specific antibodies or fragments. In certain embodiments of this aspect, the MHC protein is an MHC class I protein (e.g., HLA-A2). In certain embodiments of this aspect, the neoepitope is an NPM1c neoepitope. In one particular embodiment, the neoepitope:MHC complex is NPM1c:MHC class I (e.g., NPM1c:HLA-A2). In some embodiments of this aspect, the neoepitope is any NPM1c epitope referenced in this disclosure (e.g., an epitope having the amino acid sequence of AIQDLCLAV (SEQ ID NO: 1)). In one particular embodiment, the neoepitope:MHC complex is AIQDLCLAV:HLA-A2. In some embodiments, antibodies or antigen-binding fragments specific for the neoepitope:MHC complex are obtained using any four, five, six, seven, eight, or all of the selection steps described in Appendix 1 below. In some embodiments, instead of yeast surface display or phage surface display methods and libraries, bacterial display, eukaryotic virus display, mammalian cell display, or cell-free (e.g., ribosome display) antibody screening technology is used. Yeast surface display methods and libraries are known in the art (see, for example, Chao et al., 2006, Nature Protocols 1(2):755-768). Phage display methods and libraries are known in the art. Merz et al. (1995) J Neurosci Methods 62(1-2):213-9; Di Niro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) Methods Mol Biol 51:355-376.

[0078] antibody In one aspect, provided herein are antibodies and antigen-binding fragments thereof that bind (e.g., specifically bind) to an antigen comprising a neoepitope (e.g., a cancer neoepitope) complexed with (or presented by) an MHC protein (e.g., an MHC class I protein). In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to an antigen comprising a neoepitope (e.g., a cancer neoepitope) complexed with (or presented by) an MHC protein (e.g., an MHC class I protein) obtained by one of the methods described herein (e.g., using multiple selection steps to identify specific binders by yeast surface display, phage display, etc.; optionally using a dimeric neoepitope-MHC complex as an antigen; e.g., via immunizing a subject with the neoepitope-MHC complex or the dimeric neoepitope-MHC complex as an antigen to elicit antibody production). In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to an antigen comprising a cancer neoepitope complexed with (or presented by) an MHC class I protein (e.g., a protein encoded by HLA-A2 or the HLA-A*02 allele group), obtained by one of the methods described herein. In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that do not bind, or do not substantially bind, to an MHC protein alone. In one aspect, provided herein are antibodies or antigen-binding fragments thereof that do not bind, or do not substantially bind, to a control peptide complexed with an MHC protein. In one aspect, provided herein are antibodies or antigen-binding fragments thereof that do not bind, or do not substantially bind, to a neoepitope alone (without an MHC protein).

[0079] Functional MHC class I molecules contain an α heavy chain and a β2-microglobulin chain. Peptide binding by MHC class I molecules is achieved by interactions between peptide amino acid side chains and discrete pockets within the peptide-binding groove of the MHC molecule formed by the α1 and α2 domains of the heavy chain. Typically, for human leukocyte antigens (HLA), the primary binding energy comes from interactions between residues at position 2 and the C-terminus of the peptide with the B and F binding pockets of the MHC molecule, respectively, although side chains throughout the peptide can enhance or diminish MHC binding capacity (see, e.g., Guo, et al (1992) Nature 360:364; Silver et al (1992) Nature 360:367; Gorga et al (1992) Proteins 12;87; Madden (1995) Annu Rev Immunol 13:587; Madden et al (1993) Cell 75;693; Madden et al (1992) Cell 70:1035; Bjorkman, et al (1987) Nature 329:512; Saper et al (1991) J Mol Biol 219:277). In the case of a 9 amino acid residue peptide, the C-terminal residue (position 9) interacts with the F binding pocket of the MHC molecule.

[0080] MHC molecules are highly polymorphic, with thousands of allelic variants identified at class IA and B loci. Most of the polymorphism occurs in the peptide-binding pocket, resulting in MHC molecules with a wide range of peptide-binding specificities. Despite this polymorphism, it is known in the art that HLA class I molecules can be grouped into several groups (i.e., supertypes) based on common peptide-binding specificities. Each group (supertype) is defined by a peptide consensus sequence that reflects the position of the "anchor residues," or residues critical for MHC binding, in the peptide. For example, HLA class I molecules of the A2-supertype (i.e., HLA-A2, or proteins encoded by the HLA-A*02 allele group) share common specific binding for peptides with a small aliphatic residue (e.g., alanine, tyrosine, serine, valine, leucine, isoleucine, methionine, glutamine) at position 2 of the peptide, as well as an aliphatic residue (e.g., leucine, isoleucine, valine, methionine) or a small hydrophobic residue (e.g., alanine, valine) at the C-terminus (see, e.g., Sidney, et al (2008) BMC Immunology 9:1).

[0081] In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof that bind (e.g., specifically bind) to an antigen comprising an acute myeloid leukemia (AML)-associated mutant nucleophosmin protein neoepitope complexed with (or presented by) an MHC class I protein, e.g., HLA-A2 (e.g., NPM1c:HLA-A2).

[0082] Genomic analyses of AML have shown that it has a lower mutational burden than most other adult cancers, with an average of 13 coding mutations per AML patient (see Ley et al., N Engl J Med 368: 2059 (2013); Alexandrov et al., NATURE 500: 415 (2013); Kandoth et al., NATURE 502 333 (2013)). However, somatic mutations in AML often reside in the same gene (see Ley et al., N Engl J Med 368: 2059 (2013); Papaemmanuil et al., N Engl J Med 374: 2209 (2016)). Therefore, neoantigens derived from these hotspot mutations represent attractive targets for tumor-specific immunotherapy (see van der Lee et al., J CLIN INVEST 129: 774 (2019)). One of the most common mutations is a four-nucleotide duplication in the critical driver gene encoding nucleophosmin (NPM1; encoded by NPM1), which is present in 30–35% of adult AML patients overall (see Ley et al., N Engl J Med 368: 2059 (2013); Papaemmanuil et al., N Engl J Med 374: 2209 (2016); Falini et al., N Engl J Med 352: 254 (2005)). Such mutations in NPM1 result in its abnormal cytoplasmic localization, and this mutant protein is designated NPM1c. AML-associated NPM1c mutant proteins are HLA class I-restricted and generate leukemic neoantigens that are presented on leukemic blasts from patients with the HLA-A*02:01 allele and several other alleles. For example, NPM1c produces a leukemia-specific neoantigen epitope (AIQDLCLAV (SEQ ID NO: 1), abbreviated as AIQ) that is presented by the most common HLA-A*0201 allele (approximately 50% of the human population) (see Greiner et al., BLOOD 120: 1282 (2012)).

[0083] In some embodiments, provided herein are antibodies and antigen-binding fragments thereof that bind to an antigen comprising an NPM1c neoepitope complexed with (or presented by) an MHC class I protein, e.g., HLA-A2. The length of the NPM1c neoepitope can be any length that is reasonable for a peptide to bind to an MHC class I molecule. In some embodiments, the length of the NPM1c neoepitope is 5-20 amino acids, 6-19 amino acids, 7-18 amino acids, 8-17 amino acids, 8-16 amino acids, 8-15 amino acids, 8-15 amino acids, 8-14 amino acids, 8-13 amino acids, 8-12 amino acids, 9-12 amino acids, or 9-11 amino acids. In some embodiments, the length of the NPM1c neoepitope is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids. In some aspects, the NPM1c neoepitope is 12 amino acids in length. In some embodiments, the NPM1c neoepitope is 11 amino acids in length. In some aspects, the NPM1c neoepitope is 10 amino acids in length. In some aspects, the NPM1c neoepitope is 9 amino acids in length. In some aspects, the NPM1c neoepitope is 8 amino acids in length. In some embodiments, the NPM1c neoepitope is a peptide of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 consecutive amino acids within a polypeptide that is 10, 15, 20, 30, 40, 50, or 100 amino acid residues in length.

[0084] In some embodiments, the NPM1c neoepitope binds to an MHC class I protein that is HLA-A2. In some embodiments, the NPM1c neoepitope that binds to HLA-A2 comprises an amino acid sequence in which position 2 of the amino acid sequence is a small aliphatic residue (e.g., alanine, tyrosine, serine, valine, leucine, isoleucine, methionine, glutamine) and the C-terminal residue of the amino acid sequence is an aliphatic residue (e.g., leucine, isoleucine, valine, methionine) or a small hydrophobic residue (e.g., alanine, valine). In some embodiments, the NPM1c neoepitope that binds to HLA-A2 comprises an amino acid sequence in which position 2 of the amino acid sequence is valine, isoleucine, or leucine and the C-terminal residue of the amino acid sequence is valine, leucine, or isoleucine. In some embodiments, where the NPM1c neoepitope is 8 amino acid residues in length, the C-terminal amino acid is at position 8. In some embodiments, where the NPM1c neoepitope is 9 amino acid residues in length, the C-terminal amino acid is at position 9. In some embodiments, where the NPM1c neoepitope is 10 amino acid residues in length, the C-terminal amino acid is at position 10. In some embodiments, where the NPM1c neoepitope is 11 amino acid residues in length, the C-terminal amino acid is at position 11. In some embodiments, where the NPM1c neoepitope is 12 amino acid residues in length, the C-terminal amino acid is at position 12.

[0085] Neoepitopes derived from NPM1c that bind to HLA-A2 are known in the art. For example, Greiner (2012) Blood 120:1282 identifies the amino acid sequences of 9-mer NPM1c neoepitopes that bind to HLA-A2, including AIQDLCLAV (SEQ ID NO: 1) and AIQDLCVAV (SEQ ID NO: 71). As a further example, van der Lee (2019) J Clin Invest 129:774 identifies the amino acid sequences of NPM1c neoepitopes that bind to HLA-A2 class I molecules, including CLAVEEVSL (SEQ ID NO: 72), as well as the amino acid sequences of NPM1c neoepitopes that bind to MHC class I molecules encoded by other HLA haplotypes, including VEEVSLRK (SEQ ID NO: 73), AVEEVSLR (SEQ ID NO: 74), AVEEVSLRK (SEQ ID NO: 75), and CLAVEEVSLRK (SEQ ID NO: 76).

[0086] In some embodiments, provided herein are antibodies and antigen-binding fragments thereof that bind (e.g., specifically bind) to an antigen comprising a neoepitope of a mutant nucleophosmin protein complexed with (or presented by) an MHC class I protein (e.g., NPM1c:HLA-A2), where the mutation in the nucleophosmin protein results from a four-nucleotide duplication in the gene encoding nucleophosmin. In some embodiments, provided herein are antibodies and antigen-binding fragments thereof that bind (e.g., specifically bind) to an antigen comprising a cytoplasmic (located in the cytoplasm) mutant nucleophosmin protein neoepitope complexed with (or presented by) an MHC class I protein (e.g., NPM1c:HLA-A2). In some embodiments, the neoepitope is an 8-, 9-, 10-, 11-, or 12-amino acid peptide derived from the mutant nucleophosmin protein. In some embodiments, the neoepitope is an 8, 9, 10, 11, or 12 amino acid peptide derived from 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues of the mutant nucleophosmin protein. In some embodiments, the neoepitope is an 8, 9, 10, 11, or 12 amino acid peptide derived from 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues at the C-terminus of the mutant nucleophosmin protein. In some embodiments, the mutant nucleophosmin protein comprises the amino acid sequence set forth in SEQ ID NO:56. In some embodiments, the mutant nucleophosmin protein comprises an amino acid sequence having one or more mutations (e.g., insertions, deletions, substitutions) relative to the amino acid sequence of wild-type nucleophosmin (e.g., SEQ ID NO: 54). In some embodiments, the mutant nucleophosmin protein neoepitope is an 8, 9, 10, 11, or 12 amino acid peptide derived from a protein comprising the amino acid sequence of SEQ ID NO: 56.In some embodiments, the mutant nucleophosmin protein neoepitope is an 8, 9, 10, 11, or 12 amino acid peptide derived from a protein comprising an amino acid sequence having one or more mutations (e.g., insertions, deletions, substitutions) relative to the amino acid sequence of wild-type nucleophosmin (e.g., SEQ ID NO: 54). In some embodiments, the mutant nucleophosmin protein neoepitope is an 8, 9, 10, 11, or 12 amino acid peptide derived from 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the mutant nucleophosmin protein neoepitope is an 8, 9, 10, 11, or 12 amino acid peptide derived from a protein having an amino acid sequence with one or more mutations (e.g., insertions, deletions, substitutions) relative to the amino acid sequence of wild-type nucleophosmin (e.g., SEQ ID NO: 54), wherein the one or more mutations are within a region of the protein that is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length, and the neoepitope is derived from the region of the protein containing the one or more mutations. In some embodiments, the mutant nucleophosmin protein neoepitope is an 8, 9, 10, 11, or 12 amino acid peptide derived from 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues at the C-terminus of a protein having the amino acid sequence set forth in SEQ ID NO:56.In some embodiments, the mutant nucleophosmin protein neoepitope is an 8, 9, 10, 11, or 12 amino acid peptide derived from a protein having an amino acid sequence with one or more mutations (e.g., insertions, deletions, substitutions) relative to the amino acid sequence of wild-type nucleophosmin (e.g., SEQ ID NO: 54), wherein the one or more mutations are within a region of the protein proximal to the C-terminus (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues from the C-terminus), and the neoepitope is derived from a region of the protein containing the one or more mutations. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to an antigen comprising a neoepitope of a protein comprising the amino acid sequence of SEQ ID NO: 56 complexed with (or presented by) an MHC class I protein (e.g., an HLA-A2 protein).

[0087] In some embodiments, the mutant nucleophosmin protein comprises the C-terminal amino acid sequence MTDQEAIQDLCLAVEEVSLRK (SEQ ID NO: 57). In some embodiments, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to an antigen comprising a neoepitope of NPM1c protein comprising the C-terminal amino acid sequence MTDQEAIQDLCLAVEEVSLRK (SEQ ID NO: 57) complexed with (or presented by) an MHC class I protein (e.g., an HLA-A2 protein). In some embodiments, the neoepitope is an 8-, 9-, 10-, 11-, or 12-amino acid peptide derived from the C-terminal amino acid sequence MTDQEAIQDLCLAVEEVSLRK (SEQ ID NO: 57) of NPM1c protein. In some embodiments, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to an antigen comprising an NPM1c neoepitope complexed with (or presented by) an HLA-A2 protein or a protein encoded by the HLA-A*02 allele group (i.e., NPM1c:HLA-A2). In some embodiments, the NPM1c is human NPM1c.

[0088] In some aspects, provided herein is an antibody or antigen-binding fragment thereof that binds (e.g., specifically binds) to an antigen comprising a cytoplasmic variant nucleophosmin protein neoepitope complexed with (or presented by) an MHC class I protein (e.g., an HLA-A2 protein or a protein encoded by the HLA-A*02 allele group), wherein the amino acid sequence of the neoepitope comprises AIQDLCLAV (SEQ ID NO: 1), AIQDLCVAV (SEQ ID NO: 71), CLAVEEVSL (SEQ ID NO: 72), VEEVSLRK (SEQ ID NO: 73), AVEEVSLR (SEQ ID NO: 74), AVEEVSLRK (SEQ ID NO: 75), or CLAVEEVSLRK (SEQ ID NO: 76). In some aspects, provided herein are antibodies and antigen-binding fragments thereof that bind to an antigen presented by HLA-A2 comprising an amino acid sequence selected from AIQDLCLAV (SEQ ID NO: 1), AIQDLCVAV (SEQ ID NO: 71), CLAVEEVSL (SEQ ID NO: 72), VEEVSLRK (SEQ ID NO: 73), AVEEVSLR (SEQ ID NO: 74), AVEEVSLRK (SEQ ID NO: 75), and CLAVEEVSLRK (SEQ ID NO: 76). In some aspects, provided herein are antibodies and antigen-binding fragments thereof that bind to an antigen presented by HLA-A2 comprising the amino acid sequence AIQDLCLAV (SEQ ID NO: 1).

[0089] In some aspects, the antibodies or antigen-binding fragments thereof described herein do not bind or do not substantially bind to an MHC class I protein alone and / or a control peptide complexed with an MHC class I protein (e.g., the control peptide has the same number of amino acids as the neoepitope but is derived from a different protein than the protein from which the neoepitope is derived).

[0090] In some aspects, the antibodies or antigen-binding fragments thereof described herein do not bind or do not substantially bind to the cytoplasmic mutant nucleophosmin protein neoepitope alone (without an MHC class I protein, e.g., HLA-A2).

[0091] In some embodiments, the NPM1c neoepitope comprises the amino acid sequence AIQDLCLAV (SEQ ID NO: 1), and the MHC class I protein is an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele). In some embodiments, the NPM1c neoepitope comprises an amino acid sequence selected from AIQDLCVAV (SEQ ID NO: 71), CLAVEEVSL (SEQ ID NO: 72), VEEVSLRK (SEQ ID NO: 73), AVEEVSLR (SEQ ID NO: 74), AVEEVSLRK (SEQ ID NO: 75), and CLAVEEVSLRK (SEQ ID NO: 76), and the MHC class I protein is an HLA-A2 protein (e.g., a protein encoded by the HLA-A*02:01 allele).

[0092] In some aspects, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an antigen comprising a neoepitope comprising the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) complexed with a class I major histocompatibility complex (MHC class I) protein, wherein any one, two, three, four, five, or six amino acids of the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) have been substituted. In some aspects, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to an antigen comprising a neoepitope comprising the amino acid sequence AIQDLCLAV complexed with a class I major histocompatibility complex (MHC class I) protein, wherein any one, two, three, or four amino acids of the amino acid sequence AIQDLCLAV have been substituted. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some aspects, the amino acid substitutions are substitutions with amino acid residues of similar size to the existing residues in the AIQDLCLAV sequence (SEQ ID NO: 1). In some aspects, the amino acid substitutions do not affect (or do not substantially affect) the binding of the antibodies and antigen-binding fragments thereof described herein to the antigen.

[0093] In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to an antigen comprising a neoepitope comprising the amino acid sequence AIQDLCLAV complexed with a class I major histocompatibility complex (MHC class I) protein, wherein one, two, or more anchor residues of the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) have been substituted (e.g., position 2 and / or position 9 of SEQ ID NO: 1, e.g., the underlined residues of AIQDLCLAV (SEQ ID NO: 1)). In some aspects, the amino acid substitutions do not affect (or do not substantially affect) binding of the antibodies and antigen-binding fragments thereof described herein to the antigen or binding of the neoepitope to a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2). In some aspects, the amino acid residue I at position 2 of AIQDLCLAV (SEQ ID NO: 1) is substituted with amino acid residue L (leucine). In some embodiments, amino acid residue I at position 2 of AIQDLCLAV (SEQ ID NO: 1) is substituted with amino acid residue V (valine), M (methionine), tyrosine (T), serine (S), glutamine (Q), or A (alanine). In some embodiments, amino acid residue V at position 9 of AIQDLCLAV (SEQ ID NO: 1) is substituted with amino acid residue I (isoleucine), L (leucine), M (methionine), or A (alanine).

[0094] In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to an antigen comprising a neoepitope comprising the amino acid sequence AIQDLCLAV in complex with a class I major histocompatibility complex (MHC class I) protein, wherein any one, two, three, four, five, or six amino acids of the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) have been substituted, and the substitutions are conservative amino acid substitutions. In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to an antigen comprising a neoepitope comprising an amino acid sequence identified in Table 1 in complex with an MHC class I protein.

[0095] [Table 1]

[0096] In some aspects, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) complexed with a class I major histocompatibility complex (MHC class I) protein, wherein any one, two, three, four, five, or six amino acids of the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) have been substituted, and wherein the antibody or antigen-binding fragment has the same or substantially the same binding affinity for the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) complexed with an MHC class I protein. In some aspects, the present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) complexed with a class I major histocompatibility complex (MHC class I) protein, wherein any one, two, three, four, five, or six amino acids of the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) are substituted, and wherein the antibodies and antigen-binding fragments thereof specifically bind with the same or better affinity to the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) complexed with an MHC class I protein. In some aspects, the antibodies and antigen-binding fragments thereof described herein bind to the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) complexed with a class I major histocompatibility complex (MHC class I) protein, wherein any one, two, three, four, five, or six amino acids of the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) are substituted, and the antibody or antigen-binding fragment has a K of 0.1 to 100 nM (e.g., 0.1 to 50 nM, 0.1 to 25 nM, 0.1 to 15 nM). DIn some aspects, the antibodies and antigen-binding fragments thereof described herein bind to the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) complexed with a class I major histocompatibility complex (MHC class I) protein, wherein any one, two, three, four, five, or six amino acids of the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) have been substituted, and the antibodies and antigen-binding fragments thereof have a K of less than 100 nM (e.g., less than 50 nM, less than 25 nM, less than 15 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM). D Combine with.

[0097] In some aspects, provided herein are antibodies and antigen-binding fragments thereof that bind to an NPM1c epitope presented by an MHC class I protein, e.g., HLA-A2 (NPM1c:HLA-A2), and have an anti-cancer or anti-tumor effect (e.g., an in vivo anti-cancer effect, optionally wherein the cancer is AML).

[0098] In some aspects, the present disclosure provides an antibody, or antigen-binding fragment thereof, that specifically binds to an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some aspects, the neoepitope comprises an amino acid sequence comprising AIQDLCLAV (SEQ ID NO: 1). In some aspects, the MHC class I protein is encoded by an HLA-A allele comprising the HLA-A*02 allele group. In some aspects, the HLA-A allele is HLA-A*02:01.

[0099] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof having a heavy chain variable region and / or a light chain variable region described herein (e.g., having the sequence of the heavy chain variable region and / or the light chain variable region of YG1 scFv; see, e.g., the sequence section and examples). In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof having one or more complementarity-determining regions (CDRs) described herein (e.g., having the CDRs of YG1 scFv; see, e.g., the sequence section and examples). In some aspects, the antibody or antigen-binding fragment thereof that binds to NPM1c:HLA-A2 is an scFv. An exemplary amino acid sequence of an scFv that specifically binds to NPM1c:HLA-A2 is set forth in SEQ ID NO:2. In some embodiments, provided herein are scFvs having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, provided herein are scFvs having at least 75%, 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, wherein at least 95% of the differences in identity from the amino acid sequence set forth in SEQ ID NO: 2 are in the framework regions of the scFv (or not in the complementarity determining regions (CDRs)).

[0100] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a VH having the amino acid sequence SEQ ID NO:5 (amino acid sequence of the heavy chain variable region (VH) of YG1 scFv). In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a VH having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:5 (amino acid sequence of the heavy chain variable region (VH) of YG1 scFv). In some aspects, provided herein is a VH having at least 75%, 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, wherein at least 95% of the differences in identity to the amino acid sequence set forth in SEQ ID NO: 5 are in the framework regions of the VH (or not in the complementarity determining regions (CDRs)).

[0101] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a VL having the amino acid sequence SEQ ID NO:3 (amino acid sequence of the light chain variable region (VL) of YG1 scFv). In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a VL having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:3 (amino acid sequence of the light chain variable region (VL) of YG1 scFv). In some aspects, provided herein are VLs having at least 75%, 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 3, wherein at least 95% or all of the differences in identity with the amino acid sequence set forth in SEQ ID NO: 3 are in the framework regions of the VL (or not in the complementarity determining regions (CDRs)).

[0102] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof, comprising a VH having the amino acid sequence SEQ ID NO: 5 (amino acid sequence of the heavy chain variable region (VH) of YG1 scFv), and a VL having the amino acid sequence SEQ ID NO: 3 (amino acid sequence of the light chain variable region (VL) of YG1 scFv). In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof, comprising a VH having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 (the amino acid sequence of the heavy chain variable region (VH) of YG1 scFv), and a VL having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 3 (the amino acid sequence of the light chain variable region (VL) of YG1 scFv). In some aspects, provided herein are VH and VL having at least 75%, 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:3, respectively, wherein at least 95% or all of the differences in identity to the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:3 are in the framework regions of the VH and VL (and not in the complementarity determining regions (CDRs)).

[0103] The CDRs of the antibodies or antigen-binding fragments of this disclosure have been defined in various ways in the art, including by Kabat, Chothia, AbM, Contact, and IMGT.

[0104] In some embodiments, the CDRs of the antibodies of the present disclosure are defined according to the Kabat system, which is based on sequence variability (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391; Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Kabat CDR locations are determined according to methods known in the art. In one embodiment, the CDRs of the antibodies and fragments thereof described herein are determined using the Kabat system. In some embodiments, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof having one or more complementarity-determining regions (CDRs) of YG1 scFv determined using the Kabat system.

[0105] In some embodiments, the CDRs of the antibodies of the present disclosure are defined according to the Chothia system, which is based on the locations of immunoglobulin structural loop regions (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al, (1992) J Mol Biol 227: 799-817; Tramontano A et al, (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). The term "Chothia CDR" and similar terms are art-recognized and refer to antibody CDR sequences determined according to the method of Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917, herein referred to as "Chothia CDRs" (see, e.g., U.S. Pat. No. 7,709,226 and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)). The locations of Chothia CDRs are determined according to methods known in the art. In some embodiments, the CDRs of the antibodies and fragments thereof described herein are determined using the Chothia system. In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof having one or more complementarity determining regions (CDRs) of YG1 scFv, as determined using the Chothia system.

[0106] In some embodiments, the CDRs of the antibodies of the present disclosure are defined according to the AbM system, which is based on AbM hypervariable regions that represent a compromise between Kabat CDRs and Chothia structural loops, and the CDRs are determined using Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). The positions of AbM CDRs are determined according to methods known in the art. In one embodiment, the CDRs of the antibodies and fragments thereof described herein are determined using the AbM system. In some embodiments, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof having one or more complementarity-determining regions (CDRs) of YG1 scFv determined using the AbM system.

[0107] In some embodiments, the CDRs of the antibodies of the present disclosure are defined according to the IMGT system (see IMGT®, the international ImMunoGeneTics information system® website imgt.org, founder and director: Marie-Paule Lefranc, Montpellier, France; see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, both of which are incorporated herein by reference in their entireties). The locations of the IMGT CDRs are determined according to methods known in the art. In one embodiment, the CDRs of the antibodies and fragments thereof described herein are determined using the IMGT system. In some embodiments, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof having one or more complementarity-determining regions (CDRs) of YG1 scFv determined using the IMGT system.

[0108] In some embodiments, the CDRs of the antibodies of the present disclosure are defined according to the Contact system. The Contact definitions are based on the analysis of available complex crystal structures (bioinf.org.uk / abs) (see MacCallum RM et al., (1996) J Mol Biol 5: 732-745; see also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)). The positions of Contact CDRs are determined according to methods known in the art. In one embodiment, the CDRs of the antibodies and fragments thereof described herein are determined using the Contact system. In some embodiments, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof having one or more complementarity-determining regions (CDRs) of YG1 scFv determined using the Contact system.

[0109] In some aspects, provided herein is an antibody or fragment thereof that specifically binds to the NPM1c epitope presented by HLA-A2 and comprises one, two, or three VH CDRs and / or one, two, or three VL CDRs of YG1 scFv as defined according to any of the above systems. For example, in one embodiment, provided herein is an antibody or fragment thereof that specifically binds to the NPM1c epitope presented by HLA-A2 and comprises one, two, or all three VH CDRs and / or one, two, or all three VL CDRs of YG1 scFv as defined by IMGT.

[0110] As known in the art, VH and VL contain CDRs surrounded by framework regions (CDR and FR sequences appear in the following order in VH and VL: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4). Optionally, the framework regions are human framework regions.

[0111] In certain aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a VH having one, two, or all three VH CDRs of a VH having the amino acid sequence SEQ ID NO:5 (amino acid sequence of the heavy chain variable region (VH) of YG1 scFv). In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a VH having one, two, or all three VH CDRs of a heavy chain variable region (VH) having the amino acid sequence SEQ ID NO:5, as defined by IMGT.

[0112] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof, comprising a VL having one, two, or all three VL CDRs of a VL having the amino acid sequence SEQ ID NO: 3 (amino acid sequence of the light chain variable region (VL) of YG1 scFv). In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof, comprising a VL having one, two, or all three VL CDRs of a VL having the amino acid sequence SEQ ID NO: 3 (amino acid sequence of the light chain variable region (VL) of YG1 scFv), as defined by IMGT.

[0113] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof, comprising a VH having one, two or all three VH CDRs of the VH having the amino acid sequence SEQ ID NO: 5 (amino acid sequence of the heavy chain variable region (VH) of YG1 scFv), and a VL having one, two or all three VL CDRs of the VL having the amino acid sequence SEQ ID NO: 3 (amino acid sequence of the light chain variable region (VL) of YG1 scFv).

[0114] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a heavy chain variable region (VH) having a VH CDR1 of the amino acid sequence SEQ ID NO:9, a VH CDR2 of the amino acid sequence SEQ ID NO:10, and / or a VH CDR3 of the amino acid sequence SEQ ID NO:11. In some embodiments, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a heavy chain variable region (VH) having a VH CDR1 of the amino acid sequence SEQ ID NO:9, a VH CDR2 of the amino acid sequence SEQ ID NO:10, and a VH CDR3 of the amino acid sequence SEQ ID NO:11, wherein one, two, three, four, or five amino acids of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11 have been substituted. In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the amino acid substitutions are substitutions with amino acid residues of a similar size. In certain embodiments, the amino acid substitutions do not affect (or substantially affect) or improve binding of the antibodies or antigen-binding fragments described herein to the antigen.

[0115] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a VL having a light chain variable region (VL) CDR1 of amino acid SEQ ID NO:6, a VL CDR2 of amino acid sequence SEQ ID NO:7, and / or a VL CDR3 of amino acid sequence SEQ ID NO:8. In some embodiments, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising a VL having a light chain variable region (VL) CDR1 of amino acid SEQ ID NO:6, a VL CDR2 of amino acid sequence SEQ ID NO:7, and / or a VL CDR3 of amino acid sequence SEQ ID NO:8, wherein one, two, three, four, or five amino acids of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 have been substituted. In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the amino acid substitutions are substitutions with amino acid residues of a similar size. In certain embodiments, the amino acid substitutions do not affect (or do not substantially affect) or improve binding of an antibody or antigen-binding fragment described herein to an antigen.

[0116] In some aspects, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof that include a VH having a heavy chain variable region (VH) CDR1 of the amino acid sequence SEQ ID NO:9, a VH CDR2 of the amino acid sequence SEQ ID NO:10, and a VH CDR3 of the amino acid sequence SEQ ID NO:11, and / or a VL having a light chain variable region (VL) CDR1 of the amino acid sequence SEQ ID NO:6, a VL CDR2 of the amino acid sequence SEQ ID NO:7, and a VL CDR3 of the amino acid sequence SEQ ID NO:8. In certain embodiments, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof that include a VH having a heavy chain variable region (VH) CDR1 of the amino acid sequence SEQ ID NO:9, a VH CDR2 of the amino acid sequence SEQ ID NO:10, and a VH CDR3 of the amino acid sequence SEQ ID NO:11, and a VL having a light chain variable region (VL) CDR1 of the amino acid sequence SEQ ID NO:6, a VL CDR2 of the amino acid sequence SEQ ID NO:7, and a VL CDR3 of the amino acid sequence SEQ ID NO:8. In some embodiments, provided herein are anti-NPM1c:HLA-A2 antibodies and antigen-binding fragments thereof comprising the VH and VL described herein, wherein one, two, three, four or five amino acids in the VH and / or VL CDRs are substituted.

[0117] In some embodiments, one or more CDRs in the VH and / or VL regions of an antibody or fragment described herein may differ by one, two, three, four or five amino acids, as long as specific binding to NPM1c:HLA-A2 is maintained.

[0118] In some embodiments, the antibodies or fragments provided herein are affinity matured, i.e., have one or more alterations in one or more complementarity determining regions compared to the described antibodies or fragments, which alterations result in improved affinity of the antibody or fragment for the antigen compared to the described antibodies or fragments. In some embodiments, the antibodies or fragments provided herein have a Kd for an antigen (e.g., NPM1c:HLA-A2) of less than 100 nM (e.g., less than 50 nM, less than 25 nM, less than 15 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM). In some aspects, the antibodies or fragments provided herein have a Kd for an antigen (e.g., NPM1c:HLA-A2) of less than 15 nM, less than 10 nM, less than 7 nM, less than 5 nM, or less than 1 nM (e.g., 0.01 to 15 nM, 0.01 to 10 nM, 0.01 to 7 nM, 0.01 to 5 nM, 0.01 to 1 nM, 0.1 to 15 nM, 0.1 to 10 nM, 0.1 to 7 nM, 0.1 to 5 nM, 0.1 to 1 nM, 1 to 15 nM, 1 to 10 nM, 1 to 7 nM, 1 to 5 nM, 5 to 15 nM, 5 to 10 nM, or 5 to 7 nM).

[0119] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises three light chain variable region complementarity determining regions (VL CDRs 1-3) and three heavy chain variable region complementarity determining regions (VH CDRs 1-3). In some embodiments, the VH CDR1 comprises an amino acid sequence having at least 80% sequence identity, or at least 81% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity, or at least 84% sequence identity, or at least 85% sequence identity, or at least 86% sequence identity, or at least 87% sequence identity, or at least 88% sequence identity, or at least 89% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the VH CDR2 comprises an amino acid sequence having at least 80% sequence identity, or at least 81% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity, or at least 84% sequence identity, or at least 85% sequence identity, or at least 86% sequence identity, or at least 87% sequence identity, or at least 88% sequence identity, or at least 89% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.In some embodiments, the VH CDR3 comprises an amino acid sequence having at least 80% sequence identity, or at least 81% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity, or at least 84% sequence identity, or at least 85% sequence identity, or at least 86% sequence identity, or at least 87% sequence identity, or at least 88% sequence identity, or at least 89% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:11.

[0120] In some embodiments, the VL CDR1 comprises an amino acid sequence having at least 80% sequence identity, or at least 81% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity, or at least 84% sequence identity, or at least 85% sequence identity, or at least 86% sequence identity, or at least 87% sequence identity, or at least 88% sequence identity, or at least 89% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the VL CDR2 comprises an amino acid sequence having at least 80% sequence identity, or at least 81% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity, or at least 84% sequence identity, or at least 85% sequence identity, or at least 86% sequence identity, or at least 87% sequence identity, or at least 88% sequence identity, or at least 89% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.In some embodiments, the VL CDR3 comprises an amino acid sequence having at least 80% sequence identity, or at least 81% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity, or at least 84% sequence identity, or at least 85% sequence identity, or at least 86% sequence identity, or at least 87% sequence identity, or at least 88% sequence identity, or at least 89% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0121] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or fragments. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are purified antibodies or fragments. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are purified to greater than 95%, greater than 97%, greater than 98%, or greater than 99% purity, as determined, for example, by electrophoresis (e.g., by SDS-PAGE, isoelectric focusing, or capillary electrophoresis) or chromatography (e.g., by ion exchange or reverse-phase HPLC) methods (see, e.g., Flatman, et al., J. Chromotogr. 848:79-87 (2007)). In some embodiments, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or fragments that specifically bind to NPM1c:HLA-A2. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are purified antibodies or fragments that specifically bind to NPM1c:HLA-A2. In some embodiments, the NPM1c neoepitope is any one described herein. In some embodiments, the NP1M1c neoepitope is AIQDLCLAV (SEQ ID NO: 1).

[0122] In some aspects, the antibodies or antigen-binding fragments thereof described herein have an affinity for an antigen (e.g., NPM1c:MHC class I) of at least 10 -7 In certain embodiments, the antibodies or antigen-binding fragments thereof described herein have a binding affinity (Kd) of at least 10 for NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). -7 M or stronger, at least 10 -8 M or stronger, at least 10 -9In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a binding affinity (Kd) of at least about 25 nM or stronger, at least about 15 nM or stronger, or at least about 10 nM or stronger to an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). In some aspects, the antibodies or antigen-binding fragments thereof described herein have a binding affinity (Kd) for an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2) between 0.1 nM and 500 nM, 0.1 nM and 100 nM, 0.5 nM and 100 nM, 0.1 nM and 50 nM, 0.5 nM and 50 nM, 0.1 nM and 25 nM, 0.5 nM and 25 nM, 0.1 nM and 15 nM, 0.5 nM and 15 nM, 0.1 nM and 10 nM, or 0.5 nM and 10 nM (or from an earlier value to a later value), or between 1 nM and 100 nM (or any value in between). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a binding affinity (Kd) for NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2) of between about 0.1 nM and about 100 nM (or from an earlier value to a later value), or about 0.5 nM to about 100 nM. In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a binding affinity (Kd) for NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2) of between about 0.1 nM and about 50 nM (or from an earlier value to a later value), or about 0.5 nM to about 50 nM.

[0123] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a binding affinity of at least 0.5±0.02×10 to an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). 4 Ms -1In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a K on of at least 1±0.02×10 to an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). 4 Ms -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a K on of at least 2.5±0.02×10 to an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). 4 Ms -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a K on of at least 5±0.02×10 to an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). 4 Ms -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a K on of 0.5±0.02×10 to NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2) or higher. 4 Ms -1 and 50±0.02×10 4 Ms -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a K on between (or from an earlier value to a later value) of 1±0.02×10 to an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). 4 Ms -1 and 10±0.02×10 4 Ms -1 (or from the previous value to the later value).

[0124] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a 50±0.02×10 -4 s -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Koff of less than 10±0.02×10 for NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). -4s -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Koff of less than 5±0.02×10 for NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). -4 s -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Koff of less than 0.5±0.02×10 for NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). -4 s -1 and 50±0.02×10 -4 s -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Koff between 1±0.02×10 for NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). -4 s -1 and 15±0.02×10 -4 s -1 (or from an earlier value to a later value).

[0125] In some aspects, the antibodies described herein are monoclonal antibodies or antigen-binding fragments thereof. In some aspects, the antibodies described herein are humanized or human antibodies. In some aspects, human antibodies or antigen-binding fragments of human antibodies are provided herein. In some aspects, humanized antibodies or antigen-binding fragments of humanized antibodies are provided herein. In some aspects, chimeric antibodies or antigen-binding fragments of chimeric antibodies (chimeric antibodies are antibodies having variable regions of one species and constant regions of another species) are provided herein.

[0126] The antibodies provided herein include immunoglobulin molecules that specifically bind to an antigen (e.g., NPM1c:HLA-A2), and immunologically active fragments of such molecules that bind to the same or substantially the same epitope of the antigen as the antibody. In some aspects, the antigen bound by the antibody, or its antigen-binding fragment, is presented by an MHC class I molecule (e.g., HLA-A2) on the surface of a cancer cell. In some embodiments, the cancer cell is an AML cell.

[0127] In some embodiments, the anti-NPM1c:HLA-A2 antibody is a human or humanized antibody or immunoglobulin comprising the three VH CDRs and / or three VL CDRs described herein, human or human-derived framework regions, and human or human-derived constant regions. Non-limiting examples of human framework regions are described in the art, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Sims et al. J. Immunol. 151:2296 (1993); Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al. J. Immunol., 151:2623 (1993); Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008); Baca et al., J. Biol. Chem. 272: 10678-10684 (1997); Rosok et al., J. Biol. Chem. 271: :22611-22618 (1996); Chothia et al., J. Mol. Biol. 278:457-479 (1998). Preferably, one or more amino acid substitutions can be made in the framework regions to improve binding of the antibodies described herein to NPM1c:HLA-A2.

[0128] In some aspects where the antibody is an immunoglobulin, antibody types that may be used include, but are not limited to, IgG, IgE, IgM, IgD, IgA, and IgY. Antibody classes that may be used include, but are not limited to, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some aspects, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody or an IgG4 antibody. In some aspects, the antibody comprises a wild-type IgG1 heavy chain constant region. In some aspects, the antibody comprises a wild-type IgG4 heavy chain constant region. In some aspects, the antibody comprises a mutant IgG1 heavy chain constant region. In some aspects, the antibody comprises a mutant IgG4 heavy chain constant region. In some aspects, the mutant IgG4 heavy chain constant region comprises any one of the following substitutions according to EU numbering: S228P, L235E, L235A, or a combination thereof. In some aspects, the antibody comprises an Fc domain comprising at least one mutation.

[0129] In some embodiments, single-chain antibodies, e.g., single-chain Fvs (scFvs), are provided herein. In some embodiments, the scFvs are human or humanized scFvs. In some embodiments, the scFvs comprise a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is a Gly-Ser linker. In some embodiments, the Gly-Ser linker is selected from the group consisting of (Gly4Ser)1 (SEQ ID NO: 58), (Gly4Ser)2 (SEQ ID NO: 59), (Gly4Ser)3 (SEQ ID NO: 60), and (Gly4Ser)4 (SEQ ID NO: 61). In some embodiments, the Gly-Ser linker comprises the amino acid sequence SGSSGGSSSG (SEQ ID NO: 4). In some aspects, provided herein is an antigen-binding fragment of an antibody, which fragment may be, but is not limited to, an Fv fragment, a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, or a disulfide-linked Fv (sdFv). In one embodiment, an Fv fragment is provided herein. In one embodiment, an Fab fragment is provided herein. In one embodiment, an F(ab')2 fragment is provided herein. In one embodiment, an F(ab')2 fragment is provided herein.

[0130] In some aspects, the antibodies or antigen-binding fragments thereof described herein can induce cytotoxicity against cancer cells targeted by such antibodies or fragments, and the cytotoxicity can be due to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or the cytotoxicity of a toxin or drug conjugated to such antibodies or fragments. In some embodiments, the antibodies or antigen-binding fragments thereof described herein have ADCC, ADCP, and CDC. In some aspects, the antibodies or antigen-binding fragments thereof described herein have ADCC and ADCP. In some aspects, the antibodies or antigen-binding fragments thereof described herein have only ADCC activity or only CDC activity.

[0131] In one embodiment, the antibody or antigen-binding fragment thereof described herein mediates antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). Methods for producing antibodies with ADCC and / or ADCP function are known in the art. Generally, the Fc region of an antibody mediates its binding to Fc receptors on neutrophils, macrophages, NK cells, eosinophils, and mast cells, leading to ADCC, and its binding to Fc receptors on macrophages, neutrophils, and dendritic cells, leading to ADCP. In some aspects, provided herein are antibodies with ADCC activity, wherein the Fc region of the antibody is of human IgG or IgE type. In one embodiment, the Fc region of a contemplated antibody is of the IgG1 isotype. In one embodiment, the Fc region of a contemplated antibody is of the IgG2 isotype. In one embodiment, the Fc region of a contemplated antibody is of the IgG3 isotype. An antibody can be bioengineered (e.g., by mutation, cross-linking, disulfide bond formation, or oligosaccharide addition) to increase its ADCC and / or ADCP activity (see, e.g., Natsume et al., 2009, Drug Des Devel Ther. 3:7-16, incorporated herein by reference). In one embodiment, the CH2 and / or CH3 domains of the Fc region of the antibody are modified at their glycosylation sites to reduce or remove fucose residues for the purpose of improving ADCC and / or ADCP activity (see, e.g., Liu et al., 2015, Ca Immunol. Res. 3:173-183; Satoh et al., 2006, Expert Opin Biol. Ther. 6:1161-1173, both of which are incorporated herein by reference). In one embodiment, the Fc region of a human IgG1 isotype is mutated by alanine substitution at position 333 of the CH2 domain. In one embodiment, the Fc region of the human IgG1 isotype is mutated at the following residues: S239D, I332E, and A330L (see, e.g., Lazar et al., 2006, PNAS 103:4005-4010).(This is incorporated herein by reference.) In one embodiment, the Fc region of the human IgG1 isotype is mutated to the following residues: S239D, I332E, and G236A (see, e.g., Richards et al., 2008, Mol. Cancer Ther. 7:2517-27). In one embodiment, the Fc region comprises amino acid substitutions at positions 298, 333, and / or 334 (EU numbering), which result in improved ADCC activity.

[0132] In one embodiment, the antibody or antigen-binding fragment thereof described herein mediates complement-dependent cytotoxicity (CDC). Methods for producing antibodies with CDC function are known in the art. In some aspects, provided herein are antibodies with CDC activity, wherein the Fc region of the antibody is of human IgG or IgM type. In one embodiment, the Fc region of the antibody is of the IgG1 isotype. In one embodiment, the Fc region of the antibody is of the IgG2 isotype. In one embodiment, the Fc region of the antibody is of the IgG3 isotype. An antibody can be bioengineered (e.g., mutated) to increase its CDC activity (see, e.g., Moore et al., 2010, MAbs 2(2):181-189; Idusogie et al., 2001, J Immunol. 166(4):2571-5; Natsume et al., 2009, Drug Des Devel Ther. 3:7-16; all of which are incorporated herein by reference). In one embodiment, an antibody having an IgG Fc is bioengineered in its Fc region to change the N-glycan structure at its glycosylation site to a G0 glycan type terminating in N-acetylglucosamine without fucose or sialic acid residues. In one embodiment, the Fc region is modified to improve CDC activity, e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, or Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0133] In one embodiment, an antibody or antigen-binding fragment thereof described herein is conjugated to a cytotoxic agent (e.g., a toxin or drug). A cytotoxic agent can be an agent that induces cell death or inhibits a vital cellular function. A cytotoxic agent can be, but is not limited to, a chemotherapeutic agent, a growth inhibitory agent, a radioisotope, or a toxin. In one embodiment, an antibody or antigen-binding fragment described herein is conjugated (e.g., conjugated to) a toxin (e.g., diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, dianthin protein, momordica charantia inhibitor, crotin, gelonin, neomycin, a trichothecene, phenomycin, mitogelin, restrictocin, sapaonaria officinalis inhibitor, curcin, Phytolaca americana protein, Aleurites fordii protein, or alpha-sarcin). In one embodiment, the antibodies or antigen-binding fragments described herein are radioisotopes (e.g., P 32 , I 131 , I 125 , At 211 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , or Pb 212) is coupled to (e.g., conjugated with) the antibody or antigen-binding fragment described herein. In one embodiment, the antibody or antigen-binding fragment described herein is coupled to (e.g., conjugated with) a drug (e.g., an anti-metabolite, an antifolate, an anthracycline (e.g., doxorubicin), methotrexate, a taxane (e.g., docetaxel), paclitaxel, an auristatin, a dolastatin, a maytansinoid, or a calicheamicin). Methods for producing antibody-drug conjugates are known in the art, including drugs that may be used in such conjugates and linkers that may be used to link the antibody to the drug (see, e.g., Peters & Brown, 2015, Biosci. Rep. 35, e00225, doi:10.1042 / BSR20150089).

[0134] Also included within the scope of the present disclosure are antibodies or fragments thereof that bind to the same epitope, and / or antibodies or fragments thereof that compete with any of the antibodies and fragments described herein (e.g., antibodies or antibody fragments comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 5 and 3, respectively) for binding to human NMP1c:HLA-A2. Antibodies and fragments thereof that recognize the same epitope or compete for binding can be identified using conventional techniques. Such techniques include, for example, immunoassays that show the ability of one antibody to block the binding of another antibody to a target antigen, i.e., competitive binding assays. Competitive binding is determined in an assay in which a test antibody inhibits the specific binding of a reference antibody to a common antigen, e.g., NMP1c:HLA-A2. Numerous types of competitive binding assays are known, including, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using I-125 labels (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 (1986)); (1990)); and direct label RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).Typically, such assays involve the use of purified antigen (e.g., NPM1c:HLA-A2) or cells bearing either of these, bound to a solid surface, an unlabeled test antibody, and a labeled reference antibody (e.g., an antibody comprising the VH and VL amino acid sequences set forth by SEQ ID NOS:5 and 3, respectively). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antibody. The test antibody is usually present in excess. Typically, when a competing antibody is present in excess, it inhibits specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Other techniques include, for example, epitope mapping, e.g., X-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of the epitope. Other methods monitor antibody binding to antigen fragments or mutated versions of the antigen, with reduced binding due to altered amino acid residues within the antigen sequence often being considered an indication of epitope identity. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of a target antibody to affinity isolate specific short peptides from a combinatorial phage display peptide library. These peptides are then used as leads for defining the epitopes corresponding to the antibodies used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that have been shown to map conformationally discontinuous epitopes.

[0135] Methods for identifying neoepitopes In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that specifically binds to an antigen comprising a neoepitope complexed with (or presented by) an MHC molecule. In some embodiments, the neoepitope is a tumor-specific or cancer-specific neoepitope. In some embodiments, the MHC molecule is an MHC class I molecule.

[0136] The tumor-specific antigen or cancer-specific antigen from which the neoepitope is derived contains an altered amino acid sequence due to non-silent somatic mutation. For example, mutation-derived neoepitopes are caused by point mutations (e.g., non-synonymous mutations that result in different amino acids in the protein); read-through mutations that alter or delete stop codons, resulting in the translation of longer proteins with new tumor-specific sequences at the C-terminus; splice site mutations that result in the inclusion of introns in mature mRNA, thus resulting in unique tumor-specific protein sequences; chromosomal rearrangements (i.e., gene fusions) that produce chimeric proteins with tumor-specific sequences at the junction of two proteins; frameshift mutations or deletions that result in new open reading frames with new tumor-specific protein sequences; and translocations.

[0137] Methods for identifying tumor neoepitopes resulting from tumor-specific or cancer-specific mutations are known in the art (see, e.g., Richters, et al. (2019) Genome Medicine 11:56; Liu, et al. (2017) Cell 168:600). Such methods generally include identifying tumor-specific mutations (e.g., using deep nucleic acid or protein sequencing methods), identifying the patient's human leukocyte antigen type and predicting the corresponding major histocompatibility complex antigens present in the tumor, identifying neoepitopes (e.g., using validated peptide-MHC binding prediction algorithms or analytical methods to generate a set of candidate T cell epitopes based on mutations present in the tumor that may bind to the patient's HLA alleles), and optionally demonstrating antigen-specific T cells against the selected neoepitopes, or demonstrating that the candidate neoepitopes bind to HLA proteins on the tumor surface.

[0138] Deep nucleic acid techniques are known in the art.Any suitable sequence analysis method can be used.Such method includes, for example, the sequence analysis using traditional Sanger sequencing based on chain termination sequencing (see, for example, Sanger, et al (1977) PNAS 74:5463).As another example, the sequence analysis method can include the use of next-generation sequencing (NGS).The method of NGS is known in the art, and includes the sequencing technology based on pyrosequencing, Illumina HiSeq and MiSeq sequencing by synthesis, supported oligonucleotide ligation and detection (SOLiD), DNA nanoball sequencing, Ion Torrent sequencing, single molecule real-time (SMRT) sequencing, Helicos sequencing and Nanopore sequencing.

[0139] Nucleic acid sequencing is carried out on the whole tumor genome, tumor exome (DNA encoding protein), targeted part of genome (for example, HLA locus) or tumor transcriptome.In some embodiments, sequencing results are compared with known control set, or with the sequencing analysis carried out on normal tissue or the patient's corresponding normal tissue.One or more algorithms are used to identify the different classes of somatic mutations present in sequencing data. For example, in some embodiments, algorithms are used to detect diversity caused by single nucleotide variants (see, e.g., Cornish, et al (2015) Biomed Res Int 2015:456479; Ghoneim, et al (2014) BMC Res Notes 7:864; Kroigard, et al (2016) PLoS One 11:e0151664) and / or to detect diversity caused by indels (see, e.g., Mose, et al (2014) 30:2813-2815; Narzisi, et al (2014) Nat Methods 11:1033-1036). Furthermore, in some embodiments, the detection of the fusion of two protein-coding sequences is carried out by analyzing RNA sequencing data and / or whole genome sequencing data (see, for example, Li, et al (2011) Bioinformatics 27:1708; Scolnick, et al (2015) PLoS One 10:30128916; Zhang, et al (2016) Genome Res 26:108; Kumar, et al (2016) Wiley Interdiscip Rev RNA 7:811). Once variants in tumor DNA or RNA are detected, the impact of each variant on the amino acid sequence of the translated polypeptide is determined using computerized tools known in the art. Furthermore, several tools are available that help predict cleavage sites in translated polypeptides and identify peptides derived from MHC class I antigen processing.Non-limiting examples of such tools include NetChop20S, NetChopCterm, and ProteaSMM (see, e.g., Nielsen, et al (2005) Immunogenetics 57:33; Tenzer, et al (2005) Cell Mol Life Sci 62:1025).

[0140] Protein sequencing methods are also known in the art.In some embodiments, trotein sequencing is performed on tumor proteome.In some embodiments, protein mass spectrometry is used to identify or verify the presence of mutant peptides bound to MHC proteins on tumor cells.Peptides are acid-eluted from tumor cells or from HLA molecules immunoprecipitated from tumors, and then identified using mass spectrometry.

[0141] In some embodiments, once variant tumor-specific peptide is identified, predicting its binding to MHC class I or II molecule requires knowledge of patient's HLA haplotype.Human MHC is encoded by HLA gene complex located on chromosome 6p21.3.This locus is highly polymorphic, and more than 12,000 alleles have been determined.HLA gene is individualized, so accurate HLA haplotype determination is necessary.Methods for HLA haplotype determination are known in the art.For example, HLA haplotype determination is carried out by sequence-specific PCR amplification and Sanger sequencing or NGS-based method. Several algorithms are available for identifying HLA class I and II haplotypes based on sequencing data, such as Polysolver (Shukla, et al (2015) Nat Biotech 33:1152), HLAMiner (Warren, et al (2012) Genome Med 4:95), and OptiType (Szolek, et al (2014) Bioinformatics 30:3310).

[0142] In some embodiments, computer algorithms are used to predict putative neoepitopes, i.e., peptide sequences that undergo binding by class I or class II MHC molecules in the form of peptide-presenting complexes and are then recognized in this form by the T cell receptors of T lymphocytes. Non-limiting examples of programs useful for identifying peptides that bind to MHC class I include SMM (Nielsen, et al (2007) BMC Bioinformatics 8:238), SMMPMBEC (Kim et al (2009) BMC Bioinformatics 10:394), Pickpocket (Zhang, et al 2009) Bioinformatics 25:1293), NetMHC (Andreatta, et al 2016) Bioinformatics 32:511), NetMHCpan (Jurtz et al (2017) J Immunol 199:3360), NetMHCcons (Karosiene, et al (2012) Immunogenetics 64:177), MHCflurry (O'Donnell, et al (2018) Cell Syst 7:129), and EDGE (Bulik-Sullivan et al (2018) Nat Biotech).

[0143] In some embodiments, once putative neoepitopes are selected, they are further tested using in vitro and / or in vivo assays. In some embodiments, selected peptides are synthesized and screened in a human HLA panel to determine binding to MHC molecules encoded by different HLA haplotypes.

[0144] Methods for producing antibodies The antibodies and fragments described herein can be produced by any method known in the art.

[0145] In some embodiments, the methods described herein can include, for example, phage display techniques, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosome display) antibody screening approaches (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935; Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000) Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng 10:1303-1310; Yeung et al. (2002) Biotechnol Prog 18:212-220; Boder et al. (2000) Methods Enzymology 328:430-444; Grabherr et al. (2001) Comb Chem High Throughput Screen 4:185-192; Michael et al. (1995) Gene Ther 2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; Schaffitzel et al. (1999) J Immunol Methods 231:119-135; Chao et al., 2006, Nature Protocols 1(2):755-768; and Hanes et al. (2000) Nat Biotechnol 18:1287-1292).

[0146] Methods for identifying antibodies using various phage display methods are known in the art. In phage display, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Such phage can be used to display antigen-binding domains of antibodies expressed from repertoire or combinatorial antibody libraries (e.g., human or mouse), such as Fab, Fv, or disulfide-bond-stabilized Fv antibody fragments. The phages used in these methods are typically filamentous phages, such as fd and M13. The antigen-binding domain is expressed as a protein recombinantly fused to either the phage coat protein pIII, pVIII, or pIX. See, for example, Shi et al. (2010) JMB 397:385-396. Examples of phage display methods that can be used to produce the immunoglobulins described herein, or fragments thereof, include those described in Brinkman et al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J Immunol Methods 184:177-186; Kettleborough et al. (1994) Eur J Immunol 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994) Advances in Immunology 57:191-280; and those disclosed in PCT publications WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, and WO 95 / 20401.Suitable methods are also described, for example, in U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0147] In some embodiments, phage display antibody libraries can be generated using mRNA collected from B cells from immunized mammals. For example, spleen cell samples containing B cells can be isolated from mice immunized with the NPM1c:HLA-A2 complex as described above. mRNA can be isolated from the cells and converted into cDNA using standard molecular biology techniques. See, for example, Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Benny KC Lo (2004), supra; and Borrebaek (1995), supra. Phage display libraries are constructed using cDNAs encoding the variable regions of immunoglobulin heavy and light chain polypeptides. Methods for generating such libraries are described, for example, in Merz et al. (1995) J Neurosci Methods 62(1-2):213-9; Di Niro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) Methods Mol Biol 51:355-376.

[0148] Methods for identifying antibodies using yeast surface display are well known in the art. An example of a yeast surface display method that can be used to produce the antibodies and fragments described herein includes the method described in Chao et al., 2006, Nature Protocols 1(2):755-768.

[0149] In some embodiments, methods for producing an antibody described herein can include immunizing a subject (e.g., a non-human mammal) with a suitable immunogen. Suitable immunogens for generating any of the antibodies described herein are described herein. For example, to generate an antibody that binds to NPM1c:HLA-A2, one skilled in the art can immunize a suitable subject (e.g., a non-human mammal, such as a rat, mouse, gerbil, hamster, dog, cat, pig, goat, llama, horse, or non-human primate) with an antigen comprising the NPM1c:HLA-A2 complex, e.g., where the NPM1c neoepitope is AIQDLCLAV (SEQ ID NO: 1). A suitable subject (e.g., a non-human mammal) can be immunized with a suitable antigen, with subsequent booster immunizations sufficient to elicit antibody production by the mammal. The immunogen can be administered to the subject (e.g., a non-human mammal) with an adjuvant.

[0150] Methods for producing antibodies using hybridoma technology are well known in the art. In some embodiments, the method involves preparing a hybridoma cell line secreting a monoclonal antibody that binds to an immunogen. For example, a suitable mammal, e.g., a laboratory mouse, is immunized with the NPM1c:HLA-A2 complex as described above. Antibody-producing cells (e.g., splenic B cells) from the immunized mammal can be isolated 2 to 4 days after at least one booster immunization with the immunogen and then grown in culture for a short period before fusing with cells of a suitable myeloma cell line. The cells can be fused, for example, in the presence of a fusion promoter, e.g., vaccinia virus or polyethylene glycol. The resulting hybrid cells are cloned to select cell clones secreting the desired antibody. For example, splenocytes from a Balb / c mouse immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag 14. After fusion, the cells are grown in a suitable medium, with the addition of selective medium, e.g., HAT medium, at regular intervals to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The resulting hybrid cells are then screened for secretion of the desired antibody (e.g., an antibody that binds to NPM1c:HLA-A2).

[0151] In some embodiments, one of skill in the art can identify antibodies of interest from non-immune-based libraries, as described, for example, in U.S. Pat. No. 6,300,064 (Knappik et al.; to Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0152] In some embodiments, a combination of selection and screening can be used to identify antibodies of interest, for example, from a population of hybridoma-derived antibodies or a phage display antibody library. Suitable methods are known in the art and are described, for example, in Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al. (1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. For example, multiple phagemid vectors, each encoding a fusion protein between a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and a different antigen-binding region, can be generated using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coli). Expression of bacteriophages in bacteria may, in some embodiments, require the use of helper phage. In some embodiments, helper phage is not required (see, for example, Chasteen et al., (2006) Nucleic Acids Res 34(21):e145). The phage produced from the bacteria is recovered and then contacted with, for example, a target antigen bound (immobilized) to a solid support. Alternatively, the phage may be contacted with the antigen in solution, and then the complex is bound to a solid support.

[0153] The antibody subpopulation screened using the above method can be characterized for its specificity and binding affinity to a specific antigen (e.g., NPM1c:HLA-A2) using any immunological or biochemical method known in the art. For example, the specific binding of an antibody to NPM1c:HLA-A2 can be determined using immunological or biochemical methods, such as, but not limited to, ELISA assays, SPR assays, immunoprecipitation assays, affinity chromatography, and equilibrium dialysis, as described above. Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, RIA, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are conventional and well known in the art.

[0154] Methods for producing chimeric antibodies are well known in the art (see, e.g., Morrison, 1985, Science 229:1202-7; Oi and Morrison, 1986, BioTechniques 4:214-221; Gillies et al., 1989, J Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415).

[0155] Methods for producing humanized antibodies are well known in the art (e.g., WO 91 / 09967; Padlan, 1991, Mol Immunol 28(4 / 5): 489-498; Studnicka et al, 1994, Prot Engineering 7(6): 805-814; Roguska et al, 1994, PNAS 91: 969-973; WO 93 / 17105; Tan et al, 2002, J Immunol 169: 1119-25; Caldas et al, 2000, Protein Eng. 13(5): 353-60; Morea et al, 2000, Methods 20(3): 267-79; Baca et al, 1997, J Biol Chem 272(16):10678-84; Roguska et al, 1996, Protein Eng 9(10): 895-904; Couto et al, 1995, Cancer Res. 55 (23 Supp): 5973s-5977s; Couto et al, 1995, Cancer Res 55(8): 1717-22; Sandhu, 1994, Gene 150(2):409-10; Pedersen et al, 1994, J Mol Biol 235(3): 959-73. For example, humanized antibodies can be produced by CDR grafting.

[0156] The method for producing human antibody is well known in the art.For example, as mentioned above, human antibody can be produced by phage display or yeast surface display method using antibody library derived from human immunoglobulin sequence.Also see United States Patent (USP) 4,444,887, 4,716,111 and 5,885,793; International Publication No. 98 / 46645, International Publication No. 98 / 50433, International Publication No. 98 / 24893, International Publication No. 98 / 16654, International Publication No. 96 / 34096, International Publication No. 96 / 33735 and International Publication No. 91 / 10741. Human antibodies can also be produced using mouse-human hybridomas (see Shinmoto et al, 2004, Cytotechnology 46: 19-23; Naganawa et al, 2005, Human Antibodies 14: 27-31).

[0157] Methods for producing antibody fragments are well known in the art. For example, Fab fragments and F(ab')2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as pepsin (to produce F(ab')2 fragments) or papain (to produce Fab fragments). Methods for producing scFv fragments are also known in the art (see, for example, Ahmad et al., 2012, Clinical and Developmental Immunology, doi: 10.1155 / 2012 / 980250; Wang et al., 2006, Anal. Chem. 78, 997-1004; Pansri et al., 2009, BMC Biotechnology 9:6; Chao et al., 2006, Nature Protocols 1(2):755-768). scFvs with desired antigen-binding properties can be selected by phage display or yeast surface display technology. scFvs can be constructed by fusing the heavy and light chain variable domains of immunoglobulins via a short polypeptide linker (using recombinant expression techniques). Methods for producing single-domain antibodies (e.g., antibodies lacking light chains) are well known in the art (see, e.g., Riechmann & Muyldermans, 1999, J Immunol 231:25-38; Nuttall et al., 2000, Curr Pharm Biotechnol 1(3):253-263; Muyldermans, 2001, J Biotechnol 74(4): 277-302).

[0158] Methods for making bispecific antibodies are well known in the art (see, e.g., Konterman, 2012, MAbs 4: 182-197; Gramer et al., 2013, MAbs 5:962-973).

[0159] In embodiments where the selected CDR amino acid sequence is a short sequence (e.g., less than 10-15 amino acids in length), the nucleic acid encoding the CDR can be chemically synthesized, for example, as described in Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1):129-130 and U.S. Pat. No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDR can be replaced with a chemically synthesized nucleic acid using standard molecular biology techniques. The 5' and 3' ends of the chemically synthesized nucleic acid can be synthesized to include sticky-end restriction enzyme sites for use in cloning the nucleic acid into a nucleic acid encoding the variable region of a donor antibody.

[0160] In some embodiments, the antibodies described herein comprise an altered heavy chain constant region that has enhanced or reduced effector function (or no effector function) compared to its corresponding unaltered constant region. Effector functions associated with the constant regions of the antibodies described herein can be modulated by altering the properties of the constant region or Fc region. Altered effector functions include, for example, modulation of one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and proinflammatory responses. Modulation refers to an increase, decrease, or elimination of effector function activity exhibited by a subject antibody containing an altered constant region compared to the activity of the unaltered form of the constant region. An altered constant region with altered FcR binding affinity and / or ADCC activity and / or altered CDC activity is a polypeptide that has either enhanced or reduced FcR binding activity and / or ADCC activity and / or CDC activity compared to the unaltered form of the constant region. An altered constant region that exhibits increased binding to an FcR binds at least one FcR with higher affinity than the unaltered polypeptide, and an altered constant region that exhibits decreased binding to an FcR binds at least one FcR with lower affinity than the unaltered form of the constant region.

[0161] Methods for conferring CDC or ADCC activity to antibodies are well known in the art (see, e.g., Kellner et al., 2014, Methods 65: 105-113; WO 2012010562; Natsume et al., 2009, Drug Design, Development and Therapy 3(3):7-16). Such methods include, but are not limited to, Fc isotype shuffling, amino acid mutations in the Fc region that confer enhanced CDC and / or ADCC activity, and alterations in the glycosylation profile of the Fc region that confer enhanced CDC and / or ADCC activity.

[0162] For example, the antibodies described herein may contain altered constant regions that exhibit enhanced or reduced complement-dependent cytotoxicity (CDC). Modulated CDC activity can be achieved by introducing one or more amino acid substitutions, insertions, or deletions into the Fc region of the antibody. See, for example, U.S. Patent No. 6,194,551. Alternatively or additionally, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibodies thus generated may have improved or reduced internalization capability and / or increased or decreased complement-mediated cell killing. See, e.g., Caron et al. (1992) J Exp Med 176:1191-1195 and Shopes (1992) Immunol 148:2918-2922; PCT Publication Nos. WO 99 / 51642 and WO 94 / 29351; Duncan and Winter (1988) Nature 322:738-40; and U.S. Pat. Nos. 5,648,260 and 5,624,821.

[0163] Any of the antibodies described herein can be screened and / or tested for its ability to modulate any activity or function due to an antigen, e.g., NPM1c:HLA-A2, either in vitro or in vivo using any immunological or biochemical-based method known in the art.

[0164] The antibody or antigen-binding fragment thereof described herein can be produced using various techniques known in the art of molecular biology and protein chemistry.For example, the nucleic acid encoding one or both of the heavy and light chain polypeptides of the antibody can be inserted into an expression vector containing transcriptional and translational regulatory sequences, including, for example, a promoter sequence, a ribosome binding site, a transcriptional start and stop sequence, a translational start and stop sequence, a transcriptional terminator signal, a polyadenylation signal, and an enhancer or activator sequence.Regulatory sequences include a promoter and a transcriptional start and stop sequence.In addition, the expression vector can contain multiple replication systems so that it can be maintained in two different organisms, for example, in mammalian cells or insect cells for expression, and in a prokaryotic host for cloning and amplification.

[0165] Several potential vector systems are available for the expression of cloned heavy and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies on integration of the desired gene sequence into the host cell genome. Cells with stably integrated DNA can be selected by co-introducing a drug resistance gene, such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene can be linked to the DNA gene sequence to be expressed or can be introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements that confer autonomous replication to extrachromosomal plasmids. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyomavirus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0166] The expression vector can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction will depend primarily on the type of target cell, as described below. Exemplary methods include CaPO precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0167] Suitable host cells for expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), and primary cell lines.

[0168] In some embodiments, antibodies or fragments thereof can be expressed in and purified from transgenic animals (e.g., transgenic mammals). For example, antibodies can be produced in transgenic non-human mammals (e.g., rodents) and isolated, for example, from milk, as described in Houdebine (2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J Immunol Methods 231(1-2):147-157.

[0169] Antibodies and fragments thereof can be produced from host cells transformed with an expression vector containing nucleic acid encoding the antibody or fragment by culturing the cells under conditions sufficient to allow expression of the protein for a certain period of time. Such conditions for protein expression will vary depending on the choice of expression vector and host cell and will be easily ascertained by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning—A Laboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The selection of codons, suitable expression vectors, and suitable host cells will vary depending on several factors and can be easily optimized as needed. The antibodies (or fragments thereof) described herein can be expressed in mammalian cells or other expression systems, including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0170] After expression, antibodies and their fragments can be isolated. Antibodies or their fragments can be isolated or purified using a variety of methods known to those skilled in the art, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, antibodies can be purified using a standard anti-antibody column (e.g., a Protein A or Protein G column). Ultrafiltration and diafiltration techniques, combined with protein concentration, are also useful. See, for example, Scopes (1994) "Protein Purification, 3rd edition," Springer-Verlag, New York City, New York. The degree of purification required will vary depending on the desired use. In some cases, purification of the expressed antibody or its fragment is not necessary.

[0171] Methods for determining the yield or purity of purified antibodies or fragments thereof are known in the art and include, for example, Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis (e.g., using a protein stain such as Coomassie blue or colloidal silver stain).

[0172] An antibody or antigen-binding fragment thereof can be modified after its expression and purification. The modification can be covalent or non-covalent. Such modifications can be introduced into an antibody or fragment, for example, by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be selected using any of a variety of criteria, including, for example, structural analysis or amino acid sequence analysis of the antibody or fragment.

[0173] In some embodiments, an antibody or antigen-binding fragment thereof can be conjugated to a heterologous moiety. The heterologous moiety can be, for example, a heterologous polypeptide, a therapeutic or cytotoxic agent (e.g., a toxin or drug), or a detectable label, for example, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, for example, antigen tags (e.g., FLAG (DYKDDDDK (SEQ ID NO: 44)), polyhistidine (6-His; HHHHHH (SEQ ID NO: 45), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 46)), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibody or fragment. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detection markers, such as Luciferase. Suitable radiolabels include, for example, 32P, 33P, 14C, 125I, 131I, 35S, and 3H. Suitable fluorescent labels include fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Examples of luminescent labels include, but are not limited to, Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, for example, any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelates of diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzyme labels include, for example, alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0174] Two proteins (e.g., an antibody and a heterologous moiety) can be crosslinked using any of several known chemical crosslinkers. Examples of such crosslinkers are those that link two amino acid residues via a linkage containing a "hindered" disulfide bond. In these linkages, the disulfide bond within the crosslinking unit is protected (by hindering groups on both sides of the disulfide bond) from reduction, e.g., by the action of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), utilizes a terminal lysine on one protein and a terminal cysteine ​​on the other to form such a linkage between two proteins. Heterobifunctional reagents that crosslink via different coupling moieties on each protein can also be used. Other useful cross-linking agents include, but are not limited to, reagents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), reagents that link two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), reagents that link an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), reagents that link an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and reagents that link an amino group and a guanidinium group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0175] In some embodiments, a radiolabel can be directly conjugated to the amino acid backbone of an antibody. Alternatively, the radiolabel can be included as part of a larger molecule (e.g., meta-[I]iodophenyl-N-hydroxysuccinimide (I in [I]mIPNHS) that binds to free amino groups to form a metaiodophenyl (mIP) derivative of the relevant protein (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) or a chelator (e.g., DOTA or DTPA) that is then attached to the protein backbone. Methods for conjugating a radiolabel or larger molecule / chelator containing it to the antibodies or antigen-binding fragments described herein are known in the art. Such methods involve incubating the protein with the radiolabel under conditions (e.g., pH, salt concentration, and / or temperature) that favor binding of the radiolabel or chelator to the protein (see, e.g., U.S. Pat. No. 6,001,329).

[0176] Methods for conjugating fluorescent labels (sometimes referred to as "fluorophores") to proteins (e.g., antibodies) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysine) or sulfhydryl groups (e.g., of cysteine) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophore. In some embodiments, fluorophores can be conjugated to heterobifunctional crosslinker moieties such as sulfo-SMCC. A suitable conjugation method involves incubating an antibody protein, or a fragment thereof, with the fluorophore under conditions that promote binding of the fluorophore to the protein. See, for example, Welch and Redvanly (2003) "Handbook of Radiopharmaceuticals: Radiochemistry and Applications," John Wiley and Sons (ISBN 0471495603).

[0177] In some embodiments, the antibody or fragment can be modified with a moiety that improves the stabilization and / or retention of the antibody in circulation, for example, in blood, serum, or other tissues.For example, the antibody or fragment can be PEGylated or HESylated (Fresenius Kabi, Germany; see, for example, Pavisic et al. (2010) Int J Pharm 387(1-2):110-119), as described, for example, in Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476). The stabilizing moiety may improve the stability or retention of the antibody (or fragment) by at least about 1.5-fold (e.g., by at least about 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, or 50-fold or more).

[0178] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be glycosylated. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be subjected to enzymatic or chemical treatment or produced from cells so that the glycosylation of the antibodies or fragments is reduced or absent. Methods for producing antibodies with reduced glycosylation are known in the art and are described, for example, in U.S. Patent No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

[0179] bispecific molecules In certain embodiments, antigen-binding constructs that can be used to form bispecific molecules are provided herein. An antibody against an antigen containing a mutant nucleophosmin protein neoepitope complexed with an MHC protein (e.g., an anti-NPM1c:MHC class I antibody), or an antigen-binding fragment thereof, can be derivatized or linked to another molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. For example, an anti-NPM1c:HLA-A2 antibody, or an antigen-binding fragment thereof (e.g., scFv), can be linked to an antibody or antigen-binding fragment thereof (e.g., scFv) that specifically binds to an antigen expressed on T cells (e.g., CD3) or natural killer cells. Multispecific molecules that bind to more than two different binding sites and / or target molecules can also be created by derivatizing or linking an antibody, or an antigen-binding fragment thereof, as described herein to multiple other molecules; such multispecific molecules are also intended to be included within the scope of the term "bispecific molecule" as used herein. To generate a bispecific molecule as described herein, an antibody as described herein, or antigen-binding fragment thereof, can be linked (e.g., by chemical coupling or conjugation, genetic fusion, non-covalent association, etc.) to one or more other molecules, e.g., another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule results.

[0180] Thus, in certain embodiments, provided herein are bispecific molecules comprising at least one first binding specificity (i.e., the first antigen-binding domain of the bispecific molecule) for an antigen comprising a mutant nucleophosmin protein neoepitope complexed with an MHC protein (e.g., NPM1c:HLA-A2) and a second binding specificity (i.e., the second antigen-binding domain of the bispecific molecule) for a second target epitope. In embodiments in which the bispecific molecules described herein are multispecific, the molecules can further comprise a third binding specificity.

[0181] In certain embodiments, the specificity of the first antigen-binding domain of the bispecific molecule and the second antigen-binding domain of the bispecific molecule are the same, while in certain embodiments, the specificity of the first antigen-binding domain of the bispecific molecule and the second antigen-binding domain of the bispecific molecule are different.

[0182] In one embodiment, the bispecific molecules described herein include at least one antibody, or antibody fragment thereof, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or a single-chain Fv (scFv), or a single-chain antibody molecule. An antibody may also be a light-chain or heavy-chain dimer, or any very small fragment thereof, such as an Fv or single-chain construct, as described in Ladner et al., U.S. Patent No. 4,946,778, the contents of which are incorporated herein by reference in their entirety.

[0183] In certain embodiments, a bispecific molecule of the present disclosure is a bispecific single-chain antibody. In one embodiment, at least one of the antigen-binding domains in a bispecific molecule of the present disclosure is a single-chain fragment of the variable region of an antibody.

[0184] In certain embodiments, the antibodies or antigen-binding fragments used in the bispecific molecules described herein are human (e.g., human monoclonal antibodies). Other antibodies that may be used in the bispecific molecules described herein are murine, chimeric, or humanized antibodies (e.g., murine, chimeric, or humanized monoclonal antibodies).

[0185] The bispecific molecules described herein can be prepared by conjugating the constituent antigen-binding domains using methods known in the art, for example, each antigen-binding domain of the bispecific molecule can be generated separately and then conjugated to one another.

[0186] When the antigen-binding domain is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid ester (sulfo-SMCC) (see, for example, Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375. Preferred conjugation agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0187] When the antigen-binding specificities are antibodies, they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0188] Alternatively, both antigen-binding domains can be encoded by the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule comprises mAb and mAb, mAb and Fab, mAb and Fab', mAb and F(ab')2, mAb and Fv, mAb and scFv, Fab and F(ab')2, Fab and Fab, Fab' and Fab', F(ab')2 and F(ab')2, scFv and scFv, Fv and Fv, or ligand and Fab fusion proteins. Bispecific antibodies can also include antibodies comprising an scFv at the C-terminus of each heavy chain. The bispecific molecules described herein can be single-chain molecules comprising one single-chain antibody and one binding determinant, or single-chain bispecific molecules comprising two binding determinants. Bispecific antibodies can also include antibodies comprising an scFv at the N-terminus of each heavy chain. Bispecific antibodies can also include antibodies comprising an scFv at the N-terminus or C-terminus of each light chain. Bispecific molecules may comprise at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

[0189] Binding of the bispecific molecule to its specific target can be confirmed using art-recognized methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of protein-antibody complexes of interest by employing a labeled reagent (e.g., an antibody) that is specific for the complex of interest.

[0190] In some embodiments, a bispecific molecule of the present disclosure binds (e.g., specifically binds) to an antigen comprising a mutant nucleophosmin protein neoepitope complexed with an MHC class I protein (e.g., HLA-A2) and simultaneously binds (e.g., specifically binds) to one or more antigens on immune effector cells, such as T cells (e.g., CD3) or natural killer cells (e.g., NKp46 or CD16A). In some embodiments, a bispecific molecule of the present disclosure specifically binds to NPM1c:HLA-A2 and simultaneously binds specifically to one or more antigens on immune effector cells. In some aspects, such binding enables retargeting of immune effector cells to tumor cells (see, e.g., Chames et al., 2009, MAbs 1:539-547). Immune effector cells include, but are not limited to, T cells, natural killer cells, macrophages, neutrophils, dendritic cells, and B lymphocytes. In some aspects, the immune effector cells targeted by the bispecific antibodies described herein are T cells (e.g., CD3), natural killer cells (e.g., NKp46 or CD16A), or macrophages. In some embodiments, the bispecific molecule comprises an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to NPM1c:HLA-A2 and an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to an antigen on an immune effector cell, e.g., a T cell (e.g., CD3), natural killer cell (e.g., NKp46 or CD16A), or macrophage.

[0191] In some embodiments, a bispecific molecule of the present disclosure binds (e.g., specifically binds) to an antigen comprising a mutant nucleophosmin protein neoepitope (e.g., an NPM1c neoepitope) complexed with an MHC class I protein (e.g., HLA-A2), and simultaneously binds (e.g., specifically binds) to one or more of the following antigens: CD3, NKp46, CD16A, CD40, CD47, 4-1BB, TGF-β, LAG-3, PD-1, TIM-3, CTLA-4, OX-40, NKp30, NKG2A, NKG2D, or DNAM-1. In one embodiment, a bispecific molecule of the present disclosure comprises a second binding specificity for CD3. In one embodiment, a bispecific molecule of the present disclosure comprises a second binding specificity for NKp46. In one embodiment, a bispecific molecule of the present disclosure comprises a second binding specificity for CD16A. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for CD40. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for CD47. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for 4-1BB. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for TGF-β. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for LAG-3. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for PD-1. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for TIM-3. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for CTLA-4. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for OX-40. In one embodiment, a bispecific molecule of this disclosure comprises a second binding specificity for NKp30. In one embodiment, a bispecific molecule of the present disclosure comprises a second binding specificity for NKG2A. In one embodiment, a bispecific molecule of the present disclosure comprises a second binding specificity for NKG2D. In one embodiment, a bispecific molecule of the present disclosure comprises a second binding specificity for DNAM-1.

[0192] In some embodiments, the bispecific molecule may be a bispecific single-chain antibody. The terms "bispecific single-chain antibody" or "single-chain bispecific antibody" refer to an antibody construct resulting from linking at least two antibody variable regions in a single polypeptide chain, lacking the constant and / or Fc portions present in intact immunoglobulins. For example, each antigen-specific portion of a bispecific single-chain antibody may be an antibody V H Region and Antibody V L Includes the area.

[0193] Advantageous variants of bispecific single chain antibodies are listed below from N- to C-terminus ("CD3" is used as an example of the second specificity, but can be replaced by another antigen, e.g., NKp46, CD16A, CD40, CD47, 4-1BB, TGF-β, LAG-3, PD-1, TIM-3, CTLA-4, OX-40, NKp30, NKG2A, NKG2D or DNAM-1): V L (NPM1c:HLA-A2)-V H (NPM1c:HLA-A2)-V H (CD3)-V L (CD3), V H (NPM1c:HLA-A2)-V L (NPM1c:HLA-A2)-V H (CD3)-V L (CD3), V L (NPM1c:HLA-A2)-V H (NPM1c:HLA-A2)-V L (CD3)-V H (CD3), V H (NPM1c:HLA-A2)-V L (NPM1c:HLA-A2)-V L (CD3)-V H (CD3), V H (CD3)-V L (CD3)-V H (NPM1c:HLA-A2)-V L (NPM1c:HLA-A2), VH (CD3)-V L (CD3)-V L (NPM1c:HLA-A2)-V H (NPM1c:HLA-A2), V L (CD3)-V H (CD3)-V H (NPM1c:HLA-A2)-V L (NPM1c:HLA-A2), or V L (CD3)-V H (CD3)-V L (NPM1c:HLA-A2)-V H (NPM1c:HLA-A2).

[0194] The antigen-binding domains of the bispecific molecules of this disclosure preferably have specificity that is at least substantially identical to the binding specificity of, for example, the antibody or immunoglobulin chain from which they are derived.

[0195] In certain embodiments, the antigen-binding domain of a bispecific molecule that binds to one or more antigens on an immune effector cell, e.g., a T cell (e.g., CD3) or a natural killer cell (e.g., NKp46 or CD16A), has at least 10 affinity to the antigen. -4 M, at least 10 -5 M, at least 10 -6 M, or at least 10 -7 In certain embodiments, the antigen-binding domain that binds to one or more antigens on immune effector cells, e.g., T cells (e.g., CD3) or natural killer cells (e.g., NKp46 or CD16A), has a binding affinity (Kd) of 10 or more for the antigen. -7 Not stronger than M (e.g., 10 -4 M and 10 -7 Between M or 10 -5 M and 10 -7 In certain embodiments, the antigen-binding domain of a bispecific molecule that binds to the NPM1c:HLA-A2 antigen has a binding affinity (Kd) of at least 10 M for the NPM1c:HLA-A2 antigen.-7 M or stronger, at least 10 -8 M or stronger, at least 10 -9 In certain embodiments, the antigen-binding domain of a bispecific molecule that binds to the NPM1c:HLA-A2 antigen has a binding affinity (Kd) of at least 20 nM or stronger, at least 15 nM or stronger, or at least 10 nM or stronger to the NPM1c:HLA-A2 antigen. In certain embodiments, the antigen-binding domain of a bispecific molecule that binds to the NPM1c:HLA-A2 antigen has a binding affinity (Kd) for the NPM1c:HLA-A2 antigen of between 0.1 nM and 500 nM, 0.1 nM and 100 nM, 0.5 nM and 100 nM, 0.1 nM and 50 nM, 0.5 nM and 50 nM, 0.1 nM and 25 nM, 0.5 nM and 25 nM, 0.1 nM and 15 nM, 0.5 nM and 15 nM, 0.1 nM and 10 nM, or 0.5 nM and 10 nM (or from an earlier value to a later value), or from 1 nM to 100 nM (or any value therebetween). Such antigen-binding domains have a binding affinity (Kd) for an antigen (e.g., CD3 antigen) on immune effector cells of at least 10 -5 M, for example, 10 -7 M, for example, 10 -8 In certain embodiments of the bispecific molecules of the disclosure, (a) the binding site of the first antigen-binding domain (which binds NPM1c:MHC class I) can have a binding affinity of at least about 10 nM or stronger, 50 nM or stronger, 25 nM or stronger, 15 nM or stronger, 10 nM or stronger. -7 M, at least about 10 -8 M, at least about 10 -9M, at least about 500 nM, at least about 100 nM, at least about 50 nM, or at least about 25 nM, or at least about 15 nM; and / or (b) the binding site of the second antigen-binding domain has an affinity of at least about 10 -7 Weaker than M, about 10 -6 Weaker than M or 10 -5 In some embodiments of the bispecific molecules of the present disclosure, (a) the binding site of the first antigen-binding domain (which binds to NPM1c:HLA-A2) has an affinity of at least about 100 nM or at least about 25 nM; and / or (b) the binding site of the second antigen-binding domain has an affinity of at least about 10 -7 Weaker than M, about 10 -6 Weaker than M or 10 -5 It has an affinity of the order of M.

[0196] According to certain embodiments mentioned above, it is advantageous for a binding site that recognizes an antigen comprising a mutant NPM1c neoepitope complexed with an MHC protein (e.g., an NPM1c:HLA-A2 antigen) to have high affinity in order to efficiently capture target cells to be destroyed. On the other hand, the binding affinity of a binding site that recognizes an antigen on an immune effector cell (e.g., a CD3 antigen) can be on the order of the binding affinity of the natural receptor for the antigen (e.g., the CD3 receptor) or the binding affinity typically found for the interaction of an immune effector cell receptor (e.g., a T cell receptor) with its ligand, i.e., an MHC-peptide complex on the surface of a target cell.

[0197] In one embodiment of the present disclosure, the first and / or second domain of the bispecific molecule of the present disclosure comprises a V domain derived from a natural antibody. H Area and V LThe antibody providing the binding site for the bispecific molecule of the present disclosure can be, for example, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, a synthetic antibody, an antibody fragment such as a Fab, Fv, or scFv fragment, or a chemically modified derivative of any of these. Monoclonal antibodies can be prepared, for example, by the technique first described by Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which involves fusing mouse myeloma cells with splenocytes from an immunized mammal with modifications developed in the art. Furthermore, antibodies or fragments thereof against the aforementioned antigens can also be obtained using the methods described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor, 1988. Antibodies can be obtained from several species, including humans. If the antibody derivatives are obtained by phage display techniques, surface plasmon resonance used in the BIAcore system can be used to increase the efficiency of phage antibodies binding to NPM1c:HLA-A2 or a second antigen (Schier, Human Antibodies Hybridomas 7 (1996), 97 105; Malmborg, J. Immunol. Methods 183 (1995), 7 13). The production of chimeric antibodies is described, for example, in WO 89 / 09622. Methods for the production of humanized antibodies are described, for example, in EP 1 0 239 400 and WO 90 / 07861. A further source of antibodies utilized in accordance with the present disclosure are so-called xenogenic antibodies.The general principles for producing xenogeneic antibodies, e.g., human antibodies in mice, are described, for example, in WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, and WO 96 / 33735. Another source of antibodies utilized in accordance with the present disclosure are human antibodies isolated and engineered using yeast surface display, as described, for example, in Chao et al., 2006, Nature Protocols 1(2):755-768.

[0198] In one embodiment, the NPM1c:HLA-A2 specific domain of the bispecific molecule described herein comprises at least one V domain that comprises or is the amino acid sequence set forth as SEQ ID NO:11 (ARLGYPTTTLLPFDY). H CDR3, at least one V comprising or having the amino acid sequence set forth as SEQ ID NO: 10 (ISGSGGST) H CDR2, and / or at least one V that comprises or is the amino acid sequence set forth as SEQ ID NO: 9 (GFTFSSYA) H Includes CDR1.

[0199] The bispecific molecules of the present disclosure comprise one or more V of the NPM1c:HLA-A2 specific domain. L Such V regions of the NPM1c:HLA-A2 specific domain may also include CDRs. L The CDR region comprises at least one V that comprises or is the amino acid sequence set forth as SEQ ID NO: 8 (QQSYSTPLT). L CDR3, at least one V comprising or having the amino acid sequence of SEQ ID NO: 7 (AAS) L CDR2, and / or at least one V that comprises or is the amino acid sequence set forth as SEQ ID NO: 6 (QSISSY) L Includes CDR1.

[0200] In one embodiment, the bispecific molecule comprises the V of an antibody that specifically binds to NPM1c:HLA-A2 in one construct (e.g., the CDRs described herein). H and / or V L It comprises CDR1, CDR2 and CDR3 of

[0201] In one embodiment, the anti-NPM1c:HLA-A2 CDRs referred to herein are according to the IMGT numbering system, which is a widely adopted standard for numbering residues in antibodies in a consistent manner (see IMGT®, the international ImMunoGeneTics information system® website imgt.org, founder and director: Marie-Paule Lefranc, Montpellier, France; see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, both of which are incorporated herein by reference in their entirety).

[0202] In certain embodiments, a bispecific molecule (e.g., a bispecific antibody or fragment) of the present disclosure simultaneously binds to NPM1c:HLA-A2 and CD3. In some embodiments, the bispecific molecule comprises a single-chain variable (scFv) fragment that binds to NPM1c:HLA-A2 and an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to CD3.

[0203] As used herein, "human CD3" refers to an antigen expressed on human T cells as part of the multimolecular T cell receptor complex, and CD3 consists of five different chains: CD3-ε, CD3-γ, CD3-δ, CD3-η, and CD3-ζ.

[0204] For example, clustering of CD3 on T cells by anti-CD3 antibodies results in T cell activation similar to antigen binding but independent of the clonal specificity of the T cell subset. Thus, a bispecific molecule that specifically binds to human CD3 at one of its antigen-binding domains can bind to the human CD3 complex expressed on human T cells and induce the elimination / lysis of target cells that carry / present the antigen (e.g., NPM1c:HLA-A2) to which the other, non-CD3-binding portion of the bispecific molecule binds. Binding of the CD3 complex by a CD3-specific binding agent (e.g., a bispecific molecule disclosed herein) results in T cell activation as known in the art; see, e.g., WO 99 / 54440 or WO 2004 / 106381. In one embodiment, the bispecific molecule of the present disclosure is advantageously capable of elimination / lysis of target cells in vivo and / or in vitro. The corresponding target cell can be a cell that expresses or presents on its surface a tumor antigen, e.g., NPM1c:HLA-A2, that is recognized by another antigen-binding domain of the bispecific molecule (i.e., the non-CD3-binding portion of the bispecific molecule). In one embodiment, the additional specificity is for NPM1c:HLA-A2.

[0205] In one embodiment of the present disclosure, the V of the CD3-specific domain of the bispecific molecule H Area and V L The region is derived from a CD3-specific antibody selected from the group consisting of OKT-3, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66, WT31, WT32, SPv-T3 b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW24B6, OKT3D, M-T301, SMC2, and F101.01. Each of these antibodies has been well described in the art (see, e.g., U.S. Pat. Nos. 8,007,796 and 884,088).

[0206] In one embodiment, the CD3-specific domain of the bispecific molecule described herein comprises at least one V H CDR3, at least one V comprising the amino acid sequence set forth as SEQ ID NO: 49 (YINPSRGYTNYNQKFKD) H CDR2, and / or at least one V comprising the amino acid sequence set forth as SEQ ID NO: 47 (GYTFTRYTMH) or SEQ ID NO: 48 (RYTMH) H Includes CDR1.

[0207] In some embodiments, the bispecific molecules of the present disclosure comprise one or more V L Such V regions of the CD3-specific domain include the CDRs. L The CDR region comprises at least one V nucleotide comprising the amino acid sequence shown as SEQ ID NO: 53 (QQWSSNPLT). L CDR3, at least one V comprising the amino acid sequence of SEQ ID NO: 52 (DTSKVAS) L CDR2, and / or at least one V comprising the amino acid sequence set forth as SEQ ID NO: 51 (RASSSVSYMN) L Includes CDR1.

[0208] In one embodiment, the bispecific molecule comprises the V of an antibody that specifically binds to CD3 in one construct (e.g., the CDRs described herein). H and / or V L It comprises CDR1, CDR2 and CDR3 of

[0209] In one embodiment, the NPM1c:HLA-A2 specific domain of the bispecific molecule comprises at least one V domain comprising the amino acid sequence set forth in SEQ ID NO: 11 (ARLGYPTTTLLPFDY). H The CD3-specific domain of the bispecific molecule comprises at least one V CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 49 (YINPSRGYTNYNQKFKD). Hat least one V comprising the CDR2 region and / or comprising the amino acid sequence set forth in SEQ ID NO: 47 (GYTFTRYTMH) or SEQ ID NO: 48 (RYTMH); H In some embodiments, such bispecific molecules comprise the V CDR1 region of each antigen-binding antibody. L For example, the NPM1c:HLA-A2 specific domain of the bispecific molecule may further comprise at least one V CDR comprising the amino acid sequence (QQSYSTPLT) set forth in SEQ ID NO:8. L CDR3 region, at least one V comprising the amino acid sequence of SEQ ID NO: 7 (AAS) L and / or the CD3-specific domain comprises at least one V comprising the amino acid sequence set forth as SEQ ID NO: 51 (RASSSVSYMN). L In one embodiment, the above-mentioned CDRs (e.g., CDR1, CDR2, CDR3) are contained in a single bispecific molecule.

[0210] In one embodiment of the present disclosure, an NPM1c:HLA-A2 and CD3 bispecific molecule comprises CDR1, CDR2, and CDR3 of both the heavy and light chains of an anti-CD3 antibody. In one embodiment of the present disclosure, an NPM1c:HLA-A2 and CD3 bispecific molecule comprises CDR1, CDR2, and CDR3 of both the heavy and light chains of an anti-NPM1c:HLA-A2 antibody. In some embodiments of the present disclosure, an NPM1c:HLA-A2 and CD3 bispecific molecule of the present disclosure comprises CDR1, CDR2, and CDR3 of both the heavy and light chains of an anti-CD3 antibody and CDR1, CDR2, and CDR3 of both the heavy and light chains of an anti-NPM1c:HLA-A2 antibody.

[0211] In one embodiment, the anti-CD3 CDRs referred to herein follow the Kabat numbering system, which is a widely adopted standard for numbering residues in antibodies in a consistent manner (Kabat et al., Sequences of Proteins of Immunological Interest, 1991, which is incorporated herein by reference in its entirety).

[0212] In one embodiment of the present disclosure, the NPM1c:HLA-A2 and CD3 bispecific molecule comprises: (a) NPM1c:HLA-A2 heavy chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO:5 H and / or an NPM1c:HLA-A2 light chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO:3 L ); and (b) a CD3 heavy chain variable region (VH) and / or light chain variable region (VL) derived from a CD3-specific antibody (e.g., an antibody selected from the group consisting of OKT-3, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, and F101.01); Includes:

[0213] In one embodiment of the present disclosure, the NPM1c:HLA-A2 and CD3 bispecific molecule comprises: (a) NPM1c:HLA-A2 heavy chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO:5 H ); and NPM1c:HLA-A2 light chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO:3 L ); and (b) CD3 heavy chain variable region (VH) and light chain variable region (VL) derived from a CD3-specific antibody (e.g., an antibody selected from the group consisting of OKT-3, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, and F101.01). Includes:

[0214] Natural killer (NK) cell activity is regulated by a complex mechanism involving both activating and inhibitory signals. Several distinct NK cell receptors have been identified that play important roles in NK cell-mediated recognition and killing of HLA class I-deficient target cells. One receptor, FcγRIIIA (CD16A), is not specific for NK cells but is involved in NK cell-mediated cytotoxicity (ADCC). Another NK cell receptor is NKp46, a member of the Ig superfamily. It is specific for NK cells, and its crosslinking induced by specific mAbs leads to strong NK cell activation, which results in an increase in intracellular Ca++ levels, induction of cytotoxicity, and lymphokine release.

[0215] In some aspects, a bispecific molecule (e.g., a bispecific antibody or fragment) of the present disclosure simultaneously binds to NPM1c:HLA-A2 and NKp46. In some embodiments, a bispecific molecule of the present disclosure simultaneously binds to NPM1c:HLA-A2 expressed or presented on tumor cells and NKp46 expressed on NK cells. In some embodiments, the bispecific molecule comprises a single-chain variable (scFv) fragment that recognizes NPM1c:HLA-A2 and an antibody or antigen-binding fragment (e.g., scFv) thereof that binds to NKp46. Antibodies and / or antigen-binding fragments that specifically bind to NKp46 are known in the art (see, e.g., WO 15 / 197593, WO 17 / 114694).

[0216] In some embodiments, a bispecific molecule (e.g., a bispecific antibody or fragment) of the present disclosure simultaneously binds to NPM1c:HLA-A2 and CD16A. In some embodiments, a bispecific molecule of the present disclosure simultaneously binds to NPM1c:HLA-A2 expressed or presented on tumor cells and CD16A expressed on NK cells. In some embodiments, the bispecific molecule comprises a single-chain variable (scFv) fragment that recognizes NPM1c:HLA-A2 and an antibody or antigen-binding fragment (e.g., scFv) thereof that binds to CD16A. Antibodies and / or antigen-binding fragments that specifically bind to CD16A are known in the art (see, e.g., Stein et al., (2012) Antibodies 1:88-123, and references cited therein).

[0217] In some embodiments, binding of a bispecific molecule provided by the present disclosure to an NK cell activates the NK cell. In some embodiments, binding of a bispecific molecule provided by the present disclosure to an NK cell induces anti-tumor activity of the NK cell. In some embodiments, binding of a bispecific antibody provided by the present disclosure to an NK cell induces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0218] Thus, in some embodiments, the present disclosure provides: (i) a first antigen-binding domain that specifically binds to an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2); and (ii) a second antigen-binding domain that specifically binds to one of the following: CD3, NKp46, CD16A, CD40, CD47, 4-1BB, TGF-β, LAG-3, PD-1, TIM-3, CTLA-4, OX-40, NKp30, NKG2A, NKG2D, or DNAM-1. and a bispecific antigen-binding polypeptide comprising:

[0219] In some embodiments, the present disclosure provides: (i) a first antigen-binding domain that specifically binds to an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2); and (ii) a second antigen-binding domain that specifically binds to CD3 (e.g., human CD3); In some embodiments, the second antigen-binding domain specifically binds to human CD3 expressed on T cells.

[0220] In some embodiments, the present disclosure provides: (i) a first antigen-binding domain that specifically binds to an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2); and (ii) a second antigen-binding domain that specifically binds to NKp46 (e.g., human NKp46); In some embodiments, the second antigen-binding domain specifically binds to human NKp46 expressed on natural killer (NK) cells.

[0221] In some embodiments, the present disclosure provides: (i) a first antigen-binding domain that specifically binds to an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2); and (ii) a second antigen-binding domain that specifically binds to CD16A (e.g., human CD16A); In some embodiments, the second antigen-binding domain specifically binds to human CD16A expressed on NK cells.

[0222] In some embodiments, the NPM1c neoepitope comprises an amino acid sequence selected from the following: AIQDLCVAV (SEQ ID NO: 71), CLAVEEVSL (SEQ ID NO: 72), VEEVSLRK (SEQ ID NO: 73), AVEEVSLR (SEQ ID NO: 74), AVEEVSLRK (SEQ ID NO: 75), and CLAVEEVSLRK (SEQ ID NO: 76). In one embodiment, the NPM1c neoepitope comprises the amino acid sequence AIQDLCLAV (SEQ ID NO: 1).

[0223] In some embodiments, the MHC class I protein is encoded by an HLA-A allele comprising the HLA-A*02 allele group. In some embodiments, the HLA-A allele is HLA-A*02:01.

[0224] In some embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:5 and the VL comprises the amino acid sequence set forth in SEQ ID NO:3.

[0225] In some embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:5, and the VL comprises an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:3.

[0226] In some embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 5 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5 and the VL comprises an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0227] In some embodiments, the first antigen-binding domain comprises the VH CDR1, VH CDR2 and VH CDR3 sequences set forth in SEQ ID NOs: 9, 10 and 11, respectively, and / or the VL CDR1, VL CDR2 and VL CDR3 sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively.

[0228] In some embodiments, the first antigen-binding domain comprises the VH CDR1, VH CDR2 and VH CDR3 sequences set forth in SEQ ID NOs: 9, 10 and 11, respectively, and the VL CDR1, VL CDR2 and VL CDR3 sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively.

[0229] In some embodiments, the first antigen-binding domain comprises an scFv, Fab, or F(ab')2, and the second antigen-binding domain comprises an scFv and Fab, or F(ab')2.

[0230] Antibodies or their corresponding immunoglobulin chains used according to the present disclosure can be further modified using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, addition, and / or recombination, and / or any other modification known in the art, either alone or in combination. Methods for introducing such modifications into the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY. The mentioned modifications are preferably made at the nucleic acid level.

[0231] Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Traditionally, recombinant production of bispecific antibodies has been based on the coexpression of two immunoglobulin heavy / light chain pairs, with the two heavy / light chain pairs having different specificities (Milstein and Cuello, (1983) Nature 305:537-539). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Fusion of the heavy chain variable region is preferably with an immunoglobulin heavy chain constant domain, including at least part of the hinge, CH2, and CH3 regions. For further details of exemplary currently known methods for generating bispecific antibodies, see, e.g., Suresh et al., (1986) Methods Enzymol. 121:210; PCT Publication No. WO 96 / 27011; Brennan et al., (1985) Science 229:81; Shalaby et al., J. Exp. Med. (1992) 175:217-225; Kostelny et al., (1992) J. Immunol. 148(5):1547-1553; Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Gruber et al., (1994) J. Immunol. 152:5368; and Tutt et al., (1991) J. Immunol. 147:60. Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0232] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been generated using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol 148(5):1547-1553. Leucine zipper peptides from Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers can be reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 provides an alternative mechanism for generating bispecific antibody fragments. These fragments contain a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for generating bispecific antibody fragments by using single-chain Fv (scFv) dimers has also been reported. See, for example, Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibody can be a "linear antibody," as described, for example, in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0233] The present disclosure also encompasses variant forms of multispecific antibodies, such as dual variable domain immunoglobulin (DVD-Ig) molecules, as described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297. DVD-Ig molecules are designed in which two different light chain variable domains (VL) from two different parent antibodies are linked in tandem by recombinant DNA technology, either directly or via a short linker, followed by a light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by a constant domain CH1 and an Fc region. Methods for producing DVD-Ig molecules from two parent antibodies are further described, for example, in PCT Publications WO 08 / 024188 and WO 07 / 024715. In some embodiments, bispecific antibodies are Fab-in-Tandem immunoglobulins in which a light chain variable region with a second specificity is fused to the heavy chain variable region of a whole antibody. Such antibodies are described, for example, in International Patent Application Publication No. WO 2015 / 103072.

[0234] Chimeric Antigen Receptor In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising an extracellular domain, wherein the extracellular domain comprises any antibody, or antigen-binding fragment thereof, or bispecific molecule described herein. In certain embodiments, the chimeric antigen receptor (CAR) provided herein comprises an extracellular domain that binds to a neoantigen (e.g., a cancer neoantigen or a tumor neoantigen). A cancer neoantigen is an antigen that is present only in cancer cells due to a mutation occurring in such cells. A cancer antigen can be expressed intracellularly and presented on the surface of cancer cells by an MHC class I protein. For example, the cancer neoantigen targeted by a CAR contemplated herein can be NPM1c:HLA-A2. In certain embodiments, an antibody or antigen-binding fragment (e.g., scFv) of the present disclosure can be used to produce a chimeric antigen receptor (CAR). In one embodiment, an antibody or antigen-binding fragment (e.g., scFv) that binds to NPM1c:HLA-A2 is used to generate a chimeric antigen receptor (CAR) polypeptide. In certain embodiments, provided herein is a chimeric antigen receptor (CAR) comprising an extracellular binding domain, wherein the extracellular binding domain comprises any antibody, or antigen-binding fragment thereof, or bispecific molecule described herein, wherein such antibody, antigen-binding fragment thereof, or bispecific molecule binds to a mutant nucleophosmin protein neoepitope (e.g., an NPM1c neoepitope) complexed with (or presented by) a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-2).

[0235] CARs are genetically engineered, artificial, membrane-bound proteins that, when expressed in immune effector cells, direct such immune effector cells to antigens and generally stimulate the immune effector cells to kill cells that present the antigen. Thus, chimeric antigen receptors (CARs) can be used to confer desired antigen specificity, e.g., anti-tumor specificity, to immune effector cells (particularly, the antigen specificity is conferred by the extracellular domain of the CAR).

[0236] CARs generally comprise an extracellular domain that binds to one or more antigens presented on cells, a transmembrane domain, and an intracellular domain that transmits an activation signal to immune effector cells when the extracellular domain binds to one or more antigens. In certain embodiments, CARs contain three domains: 1) an extracellular domain, which typically includes a signal peptide, a ligand, or an antigen recognition region (e.g., scFv), and a flexible spacer; 2) a transmembrane (TM) domain; and 3) an intracellular domain (also known as a cytoplasmic domain), which typically includes one or more signaling domains. The extracellular domain of a CAR is present on the outside of the cell and exposed to the extracellular space, making it accessible for interaction with its ligand / antigen. The TM domain allows the CAR to be anchored to the plasma membrane of the effector cell. The intracellular domain of a CAR may include one or more cytoplasmic domains derived from a signaling protein different from the protein from which the extracellular domain was derived. The intracellular domain assists in effector cell activation when the CAR binds to its ligand / antigen. In some embodiments, effector cell activation includes the induction of cytokine and chemokine production and the activation of the cytolytic activity of the effector cell. In some embodiments, the CAR redirects cytotoxicity to tumor cells.

[0237] Engagement of the antigen-binding domain of the CAR with its target antigen on the surface of the target cell results in clustering of the CAR and delivery of an activating stimulus to the CAR-containing cell. In some embodiments, a key feature of a CAR is its ability to redirect the specificity of immune effector cells, thereby inducing proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner, leveraging the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors. CARs based on scFvs engineered to contain signaling domains from CD3ζ or FcRy have been shown to deliver potent signals for T cell activation and effector function, but may not be sufficient to induce signals that promote T cell survival and proliferation in the absence of costimulatory signals. New generation CARs containing a binding domain, hinge, transmembrane domain, and signaling domain derived from CD3ζ or FcRy, along with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134, and CD278), have been shown in vitro, in animal models, and in cancer patients to increase cytokine secretion, lytic activity, survival, and proliferation in CAR-expressing T cells, in addition to directing more effective anti-tumor activity (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0238] In some aspects, provided herein are CARs that include an extracellular (antigen-binding) domain, a transmembrane domain, and an intracellular (cytoplasmic) domain that includes a cytoplasmic sequence of a CD3ζ sequence sufficient to stimulate a T cell when the antigen-binding domain binds to an antigen, and optionally, a cytoplasmic sequence of one or more (e.g., two, three, or four) costimulatory proteins (e.g., the cytoplasmic sequence of one or more of B7-H3, BTLA, CD2, CD7, CD27, CD28, CD30, CD40, CD40L, CD80, CD160, CD244, ICOS, LAG3, LFA-1, LIGHT, NKG2C, 4-1BB, OX40, PD-1, PD-L1, TIM3, 2B4, DAP10, CD137, DAP12, and a ligand that specifically binds to CD83), which result in costimulation of the T cell when the antigen-binding domain binds to the antigen. In some embodiments, the CAR may further include a linker. Further aspects of CARs and CAR-expressing immune effector cells, including exemplary extracellular (antigen-binding) domains, linkers, transmembrane domains, and intracellular (cytoplasmic) domains, are described in, e.g., Kakarla et al., Cancer J. 20:151-155, 2014; Srivastava et al., Trends Immunol. 36:494-502, 2015; Nishio et al., Oncoimmunology 4(2): e988098, 2015; Ghorashian et al., Br. J. Haematol. 169:463-478, 2015; Levine, Cancer Gene Ther. 22:79-84, 2015; Jensen et al., Curr. Opin. Immunol. 33:9-15, 2015; Singh et al., Cancer Gene Ther. 22:95-100, 2015; Li et al., Zhongguo Shi Yan Xue Ye Xue Za Zhi 22:1753-1756, 2014; Gill et al., Immunol. Rev. 263:68-89, 2015; Magee et al., Disco.Gargett et al., Front. Pharmacol. 5:235, 2014; Yuan et al., Zhongguo Shi Yan Xue Ye Xue Za Zhi 22:1137-1141, 2014; Pedgram et al., Cancer J. 20:127-133, 2014; Eshhar et al., Cancer J. 20:123-126, 2014; Ramos et al., Cancer J. 20:112-118, 2014; Maus et al., Blood 123:2625-2635, 2014; Jena et al., Curr. Hematol. Malig. Rep. 9:50-56, 2014; Maher et al., Curr. Gene Ther. 14:35-43, 2014; Riches et al., Discov. Med. 16:295-302, 2013; Cheadle et al., Immunol. Rev. 257:83-90, 2014; Davila et al., Int. J. Hematol. 99:361-371, 2014; Xu et al., Cancer Lett. 343:172-178, 2014; Kochenderfer et al., Nat. Rev. Clin. Oncol. 10:267-276, 2013; Hosing et al., Curr. Hematol. Malig. Rep. 8:60-70, 2013; Hombach et al., Curr. Mol. Med. 13:1079-1088, 2013; Xu et al., Leuk. Lymphoma 54:255-260, 2013; Gilham et al., Trends Mol. Med. 18:377-384, 2012; Lipowska-Bhalla et al., Cancer Immunol. Immunother. 61:953-962, 2012; Chmielewski et al., Cancer Immunol. Immunother. 61:1269-1277, 2013;Jena et al., Blood 116:1035-1044, 2010; Dotti et al, Immunology Reviews 257(1): 107-126, 2013; Dai et al., Journal of the National Cancer Institute 108(7): djv439, 2016; Wang and Riviere, Molecular Therapy-Oncolytics 3: 16015, 2016; U.S. Patent and Trademark Publication No. 2018 / 0057609; same as No. 2018 / 0037625; same as No. 2017 / 0362295; same as No. 2017 / 0137783; same as No. 2016 / 0152723, same as No. 2016 / 0206656, same as No. 2016 / 0199412, same as No. 2016 / 0208018, same as No. 2015 / 0232880, same as No. 2015 / 0225480 Detailed statement; same as detailed statement No. 2015 / 0224143; same as detailed statement No. 2015 / 0224142; same as detailed statement No. 2015 / 0190428; same as detailed statement No. 2015 / 0196599; same as detailed statement No. 2015 / 0152181; same as detailed statement No. 2015 / 0140023; same as detailed statement No. 2015 / 0118202; same as detailed statement No. 2015 / 0110760; same as detailed statement No. 2015 / 0099299; same as detailed statement No. 2015 / 0093822; same as detailed statement No. 2015 / 009 Item No. 3401; same as Item No. 2015 / 0051266; same as Item No. 2015 / 0050729; same as Item No. 2015 / 0024482; same as Item No. 2015 / 0023937; same as Item No. 2015 / 0017141; same as Item No. 2015 / 0017136; same as Item No. 2015 / 0017120; same as Item No. 2014 / 0370045; same as Item No. 2014 / 0370017; same as Item No. 2014 / 0369977; same as Item No. 201 Item No. 4 / 0349402; same as Item No. 2014 / 0328812; same as Item No. 2014 / 0322275; same as Item No. 2014 / 0322216; same as Item No. 2014 / 0322212; same as Item No. 2014 / 0322183; same as Item No. 2014 / 0314795; same as Item No. 2014 / 0308259; same as Item No. 2014 / 0301993; same as Item No. 2014 / 0296492; same as Item No. 2014 / 0294784;2014 / 0286973 specification; 2014 / 0274909 specification; 2014 / 0274801 specification; 2014 / 0271635 specification; 2014 / 0271582 specification; 2014 / 0271581 specification; 2014 / 0271579 specification; 2014 / 0255363 specification; 2014 / 0242701 specification; 2014 / 0242049 specification; 2014 / 0227272 specification; 2014 / 0219975 specification; 2 014 / 0170114 specification; 2014 / 0134720 specification; 2014 / 0134142 specification; 2014 / 0120622 specification; 2014 / 0120136 specification; 2014 / 0106449 specification; 2014 / 0106449 specification; 14 / 0106449 specification; 2014 / 0099340 specification; 2014 / 0086828 specification; 2014 / 0065629 specification; 2014 / 0050708 specification; 2014 / 0024809 specification; 2013 / 0344039 specification; 2013 / 0323214 specification; 2013 / 0315884 specification; 2013 / 0309258 specification; 2013 / 0288368 specification; 2013 / 0287752 specification; 2013 / 0287748 specification; 2013 / 0280221 specification; 2013 / 0280220 specification; 2013 / 0266551 specification; 2013 / 0216528 specification; 2013 / 0202622 specification; 2013 / 00 71414; 2012 / 0321667; 2012 / 0302466; 2012 / 0301448; 2012 / 0301447; 2012 / 0060230; 2011 / 0213288; 2011 / 0158957; 2011 / 0104128; 2011 / 0038836; 2007 / 0036773; and 2004 / 0043401. Further embodiments of CARs and CAR-expressing immune effector cells, including exemplary extracellular (antigen-binding) domains, linkers, transmembrane domains, and intracellular (cytoplasmic) domains, are described in WO 2016 / 168595; WO 12 / 079000;Brochure No. 2015 / 0141347; Brochure No. 2015 / 0031624; Brochure No. 2015 / 0030597; Brochure No. 2014 / 0378389; Brochure No. 2014 / 0219978; Brochure No. 2014 / 0206620; Brochure No. 2014 / 0037628; Brochure No. 2013 / 0274203; Brochure No. 2013 / 0225668 No. Brochure; No. 2013 / 0116167 Brochure; No. 2012 / 0230962 Brochure; No. 2012 / 0213783 Brochure; No. 2012 / 0093842 Brochure; No. 2012 / 0071420 Brochure; No. 2012 / 0015888 Brochure; No. 2011 / 0268754 Brochure; No. 2010 / 0297093 Brochure; No. 2010 / 0158881 pamphlet; 2010 / 0034834 pamphlet; 2010 / 0015113 pamphlet; 2009 / 0304657 pamphlet; 2004 / 0043401 pamphlet; 2014 / 0322253 pamphlet; 2015 / 0118208 pamphlet; 2015 / 0038684 pamphlet; 2014 / 0024601 pamphlet ; Brochure No. 2012 / 0148552; Brochure No. 2011 / 0223129; Brochure No. 2009 / 0257994; Brochure No. 2008 / 0160607; Brochure No. 2008 / 0003683; Brochure No. 2013 / 0121960; Brochure No. 2011 / 0052554; and Brochure No. 2010 / 0178276.

[0239] In some aspects, provided herein is a CAR comprising an intracellular domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain comprises any antibody, or antigen-binding fragment thereof, or bispecific molecule described herein. In some aspects, provided herein is a chimeric antigen receptor (CAR) comprising an intracellular domain, a transmembrane domain, and an extracellular binding domain, wherein the extracellular binding domain comprises any antibody, or antigen-binding fragment thereof, or bispecific molecule described herein, wherein such antibody, antigen-binding fragment thereof, or bispecific molecule binds to an antigen comprising an NPM1c neoepitope complexed with (or presented by) a class I major histocompatibility complex (MHC class I) protein.

[0240] In some aspects, provided herein are chimeric antigen receptors (CARs) having the intracellular, transmembrane, and / or extracellular domains of the NPM1c CAR, as described in the Examples section (see, e.g., Example 3).

[0241] In some aspects, provided herein is a CAR comprising an intracellular domain comprising one or more costimulatory domains of one or more costimulatory molecules selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, 2B4, DAP10, CD137, and DAP12. In certain embodiments, provided herein is a CAR comprising an intracellular domain comprising a CD3-zeta signaling domain and, optionally, a 4-1BB costimulatory domain. In some aspects, provided herein is a CAR comprising a transmembrane domain of CD3-zeta, CD8, CD28, NKG2D, CD16, NKp44, or NKp46. In certain embodiments, provided herein is a CAR comprising a transmembrane domain comprising a CD8 transmembrane domain. In some aspects, provided herein is a CAR comprising an extracellular domain comprising any antibody or antigen-binding fragment thereof (e.g., scFv) described herein. In certain embodiments, provided herein is a CAR comprising an extracellular domain comprising any of the antibodies described herein or antigen-binding fragments thereof (e.g., scFvs) that specifically bind to an antigen comprising a mutant nucleophosmin protein epitope (e.g., NPM1c neoepitope) complexed with (or presented by) a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2). In certain embodiments, provided herein is a CAR comprising an extracellular domain comprising any of the antibodies described herein or antigen-binding fragments thereof (e.g., scFvs) comprising a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO:5 or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:5, and the VL comprises the amino acid sequence of SEQ ID NO:3 or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:3.In certain embodiments, provided herein is a CAR comprising an extracellular domain comprising any antibody or antigen-binding fragment thereof (e.g., scFv) described herein, comprising a VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 9, a VH CDR2 having the amino acid sequence of SEQ ID NO: 10, and a CDR3 having the amino acid sequence of SEQ ID NO: 11, and / or a VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 6, a VL CDR2 having the amino acid sequence of SEQ ID NO: 7, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 8. In certain embodiments, provided herein is a CAR comprising an extracellular domain comprising any antibody or antigen-binding fragment thereof (e.g., scFv) described herein, comprising a VH comprising the VH CDRs VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequence of SEQ ID NO: 5, and / or a VL comprising the VL CDRs VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequence of SEQ ID NO: 3. In certain embodiments, provided herein is a CAR comprising an extracellular domain comprising an scFv having the amino acid sequence of SEQ ID NO:2, or an scFv having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO:2.

[0242] Examples of extracellular, transmembrane, and intracellular domains of the CARs provided herein are described below.

[0243] Antibody antigen-binding domain, including the extracellular (antigen-binding) domain of the CAR Non-limiting examples of antigen-binding domains include monoclonal antibodies (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD) (e.g., fully human or chimeric (e.g., humanized) antibodies), antigen-binding fragments of antibodies (e.g., Fab, Fab', or F(ab')2 fragments) (e.g., fragments of fully human or chimeric (e.g., humanized) antibodies), diabodies, triabodies, tetrabodies, minibodies, scFv, scFv-Fc, (scFv)2, scFab, bis-scFv, hc-IgG, BiTEs, single-domain antibodies (e.g., V-NAR domains or VhH domains), IgNAR, and multispecific (e.g., bispecific) antibodies. In one embodiment, the antigen-binding domain comprises an scFv. Methods for producing these antigen-binding domains are known in the art.

[0244] In some embodiments, the antigen-binding domain comprises at least one (e.g., one, two, three, four, five, or six) CDRs (e.g., any of the three CDRs from an immunoglobulin light chain variable domain and / or any of the three CDRs from an immunoglobulin heavy chain variable domain) of an antibody, e.g., an immunoglobulin molecule (e.g., a light chain or heavy chain immunoglobulin molecule) and an immunologically active (antigen-binding) fragment of an immunoglobulin molecule, that can specifically bind to a target antigen.

[0245] In some embodiments, the antigen binding domain is a single chain antibody (e.g., a V-NAR domain or a V H In some embodiments, the antigen-binding domain is a whole antibody molecule (e.g., a human antibody, a humanized antibody, or a chimeric antibody) or a multimeric antibody (e.g., a bispecific antibody).

[0246] In some embodiments, antigen-binding domains include antibody fragments and multispecific (e.g., bispecific) antibodies or antibody fragments. Examples of antibodies and antigen-binding fragments thereof include, but are not limited to, single-chain Fvs (scFvs), Fab fragments, Fab' fragments, F(ab')2, disulfide-linked Fvs (sdFvs), Fvs, and fragments containing either the VL or VH domains.

[0247] Additional antigen-binding domains provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly generated antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibody or antigen-binding fragment thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antigen-binding domain is an IgG1 antibody or antigen-binding fragment thereof. In some examples, the antigen-binding domain is an IgG4 antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding domain is an immunoglobulin comprising a heavy chain and a light chain.

[0248] Further examples of antigen-binding domains are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., antigen-binding fragments of human or humanized IgA, e.g., human or humanized IgA1 or IgA2), antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD), antigen-binding fragments of IgE (e.g., antigen-binding fragments of human or humanized IgE), or antigen-binding fragments of IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0249] In some embodiments, the antigen-binding domain binds to a particular antigen (e.g., a tumor-associated antigen) at a concentration of about 1×10, e.g., in saline or phosphate-buffered saline. -7 M or stronger (e.g., about 1 × 10 -8 M or stronger, approximately 1 x 10 -9 M or stronger, about 500 nM or stronger, about 100 nM or stronger, about 25 nM or stronger, about 15 nM or stronger, about 7 nM or stronger, about 5 nM or less, or about 1 nM or stronger) affinity (K D ) can be used to join them.

[0250] As will be understood by those skilled in the art, the selection of antigen-binding domains to be included in the CAR depends on the type and number of ligands that define the surface of the cells (e.g., cancer cells or tumors) to be targeted in a subject in need thereof. For example, the antigen-binding domain may be selected to recognize a tumor-specific antigen (TSA), e.g., a cancer neoantigen. For example, the tumor-specific antigen may be an NMP1c neoantigen complexed with (or presented by) an MHC class I protein (e.g., HLA-A2), e.g., NPM1c:HLA-A2. In some embodiments, the NMP1c neoantigen comprises the amino acid sequence AIQDLCLAV (SEQ ID NO: 1).

[0251] In some embodiments, the CAR molecule comprises an antigen-binding domain that recognizes a tumor antigen of acute myeloid leukemia. In some embodiments, the tumor antigen is a tumor-specific antigen (TSA), e.g., an acute myeloid leukemia neoantigen. A TSA is unique to tumor cells and is not present on other cells in the body. In one embodiment, the tumor antigen is a tumor-specific antigen. In certain embodiments, tumor-specific antigens are determined by sequencing a patient's tumor cells and identifying mutated proteins found only in tumors. These antigens are referred to as "neoantigens." Once a neoantigen is identified, therapeutic antibodies can be raised against it and used in the methods described herein. In one embodiment, the neoantigen is an NPM1c neoantigen. In one embodiment, the NMP1c neoantigen is complexed with (or presented by) an MHC class I protein (e.g., HLA-A2), e.g., NPM1c:HLA-A2.

[0252] Tumor antigens (e.g., tumor-associated antigens (TAA) and tumor-specific antigens (TSA)) that can be targeted by CAR effector cells (e.g., CAR T cells) include, but are not limited to, NPM1c:HLA-A2. In one embodiment, the tumor-specific antigen is NPM1c:HLA-A2.

[0253] Linker between the domains of CAR Provided herein are CARs that can optionally include a linker between (1) the extracellular (antigen-binding) domain and the transmembrane domain, and / or (2) the transmembrane domain and the intracellular (cytoplasmic) domain. In some embodiments, the linker can be a polypeptide linker. For example, the linker can be between about 1 amino acid and about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 4 an amino acid, or between about 2 amino acids; from about 2 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, 6 amino acids, or up to about 4 amino acids; from about 4 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, or about 6 amino acids; from about 6 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, or about 8 amino acids;From about 8 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, or about 10 amino acids; from about 10 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, or about 12 amino acids; from about 12 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 2 0 amino acids, about 18 amino acids, about 16 amino acids, or about 14 amino acids; from about 14 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, or about 16 amino acids; from about 16 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 10 0 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, or about 18 amino acids; from about 18 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, or about 20 amino acids;from about 20 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, or about 25 amino acids; from about 25 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, or up to about 30 amino acids; from about 30 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, or about 35 amino acids; from about 35 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, or about 40 amino acids; amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, or about 50 amino acids; from about 50 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, or about 60 amino acids; from about 60 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 150 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, or about 70 amino acids; about 70 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, or about 80 amino acids; about 80 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, or about 90 amino acids; about 90 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, or about 100 amino acids;It may have a length of from about 100 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, or about 200 amino acids; from about 200 amino acids to about 500 amino acids, about 400 amino acids, or about 300 amino acids; from about 300 amino acids to about 500 amino acids or about 400 amino acids; or from about 400 amino acids to about 500 amino acids;

[0254] Transmembrane domain of CAR The CARs provided herein also comprise a transmembrane domain. In some embodiments, the transmembrane domain can be derived from a natural source. In some embodiments, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains that can be used in the CARs described herein can be derived from (e.g., at least a transmembrane sequence thereof, or a portion of a transmembrane sequence thereof) the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD86, CD134, CD137, or CD154. In one embodiment, the transmembrane domain is derived from a CD4 molecule. In one embodiment, the transmembrane domain is derived from a CD8 molecule.

[0255] In some embodiments, the transmembrane domain may be synthetic. For example, in some embodiments where the transmembrane domain is derived from a synthetic source, the transmembrane domain may comprise (e.g., primarily comprise) hydrophobic residues (e.g., leucine and valine). In some embodiments, the synthetic transmembrane domain will comprise at least one (e.g., at least two, at least three, at least four, at least five, or at least six) triplets of phenylalanine, tryptophan, and valine at the end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain of the CAR may comprise a CD8 hinge domain.

[0256] In some embodiments, the transmembrane domain is naturally associated with a sequence within the cytoplasmic domain. In some embodiments, the transmembrane domain can be modified by one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions to prevent the domain from binding to other transmembrane domains (e.g., transmembrane domains of the same or different surface membrane proteins) to minimize interaction with other members of the receptor complex.

[0257] In some embodiments, the transmembrane domain of a CAR provided herein comprises the transmembrane domain of CD3-zeta, CD8, CD28, NKG2D, CD16, NKp44, or NKp46. In specific embodiments, the transmembrane domain of a CAR provided herein comprises the transmembrane domain of CD3-zeta, CD8, or CD28. In some of these embodiments, the intracellular domain of the CAR comprises a costimulatory domain of a costimulatory molecule selected from the group consisting of: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, and any combination thereof.

[0258] The intracellular (cytoplasmic) domain of CAR The intracellular domain can be any polypeptide domain known to function to transmit signals that cause the activation of immune effector cells, such as T cells, NK cells, or macrophages. Such domains or motifs can transmit the primary antigen binding signal required for the activation of T lymphocytes in response to the binding of the extracellular domain of the CAR to a target antigen. Examples of intracellular domains include, but are not limited to, ILR chains, CD28, 4-1BB, and CD3ζ.

[0259] Typically, the intracellular domain contains an ITAM (immunoreceptor tyrosine-based activation motif).

[0260] In one embodiment, the intracellular domain is or includes a CD3ζ signaling sequence (e.g., an ITAM-containing portion thereof). In one embodiment, the intracellular domain includes a lymphocyte receptor chain. In one embodiment, the intracellular domain includes a TCR / CDR3 complex protein. In one embodiment, the intracellular domain includes an Fc receptor subunit. In one embodiment, the intracellular domain includes an IL-2 receptor subunit.

[0261] The intracellular domain of a CAR provided herein can comprise two different classes of cytoplasmic signaling sequences: signaling sequences that initiate antigen-dependent activation via the TCR (primary cytoplasmic signaling sequences) (e.g., CD3ζ cytoplasmic signaling sequences), and the cytoplasmic sequences of one or more costimulatory proteins that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).

[0262] In certain embodiments, provided herein are CARs that comprise a cytoplasmic sequence of CD3ζ sufficient to stimulate a T cell when the antigen-binding domain binds to an antigen, and optionally an intracellular signaling domain that includes the cytoplasmic sequence of one or more of a costimulatory protein (e.g., one or more of CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, B7-H3, 2B4, DAP10, CD137, DAP12, a ligand that specifically binds to CD83, and any of the ITAM sequences described herein or known in the art), which results in costimulation of the T cell. In some embodiments, the entire intracellular signaling domain of the costimulatory protein is included in the intracellular domain of the CAR. In some embodiments, the intracellular domain comprises a truncated portion of the intracellular signaling domain of a costimulatory protein (e.g., a truncated portion of the intracellular signaling domain that transmits an effector function signal in a CAR-expressing immune effector cell). Non-limiting examples of intracellular signaling domains that can be included in the intracellular domain include the cytoplasmic sequences of a T cell receptor (TCR) and a co-receptor that act in concert to initiate signal transduction after antigen receptor engagement, and any variant of these sequences that contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions and has the same or nearly the same functional capability.

[0263] In some embodiments, the intracellular domain of a CAR can be designed to include a CD3ζ signaling domain, either by itself or in combination with any other desired cytoplasmic signaling sequence useful in the context of a CAR. In some embodiments, the cytoplasmic domain of a CAR can include a CD3ζ chain portion and a costimulatory cytoplasmic signaling sequence. A costimulatory cytoplasmic signaling sequence refers to a portion of a CAR that includes the cytoplasmic signaling sequence of a costimulatory protein (e.g., a ligand that specifically binds to CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83).

[0264] In some embodiments, the cytoplasmic signaling sequences in the intracellular domain of the CAR are arranged in random order. In some embodiments, the cytoplasmic signaling sequences in the intracellular domain of the CAR are linked to each other in a specific order. In some embodiments, a linker (for example, any of the linkers described herein) can be used to form a link between different cytoplasmic signaling sequences.

[0265] In some embodiments, the intracellular domain is designed to include the cytoplasmic signaling sequence of CD3ζ and the cytoplasmic signaling sequence of the costimulatory protein CD28. In some embodiments, the intracellular domain is designed to include the cytoplasmic signaling sequence of CD3ζ and the cytoplasmic signaling sequence of the costimulatory protein 4-IBB. In some embodiments, the intracellular domain is designed to include the cytoplasmic signaling sequence of CD3ζ and the cytoplasmic signaling sequences of the costimulatory proteins CD28 and 4-1BB. In some embodiments, the intracellular domain does not include the cytoplasmic signaling sequence of 4-1BB.

[0266] In some embodiments, the CAR comprises one or more costimulatory domains derived from proteins such as CD28, CD137 (also known as 4-lBB), CD134 (also known as OX40), and CD278 (also known as ICOS). In some embodiments, the CAR does not comprise a costimulatory domain derived from CD137.

[0267] In certain embodiments, the intracellular domain further comprises a cytokine. In some embodiments, the intracellular domain further comprises a self-cleaving domain (e.g., a P2A self-cleaving peptide) and a cytokine, wherein cleavage of the self-cleaving domain releases the cytokine. In some embodiments, the self-cleaving domain (e.g., a P2A self-cleaving peptide) and cytokine are disposed at the C-terminus of the CAR protein and its intracellular domain. In some embodiments, the cytokine is one or more of the following: IL-12, IL-7, IL-13, IL-15, IL-4, IL-10, TNF-α, IFN-γ, TGF-β, and CCL19. In one embodiment, the cytokine is IL-12. In one embodiment, the cytokine is IL-7. In one embodiment, the cytokine is IL-13. In one embodiment, the cytokine is IL-15. In one embodiment, the cytokine is IL-4. In one embodiment, the cytokine is IL-10. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is IFN-γ. In one embodiment, the cytokine is TGF-β. In one embodiment, the cytokine is CCL19. Immune effector cells modified to express cytokines are known in the art (see, for example, Adachi et al., 2018, Nature Biotechnology, doi:10.1038 / nbt.4086; Liu et al., 2019, J. Immunol., doi:10.4049 / jimmunol.1800033; Krenciute et al., 2017, Cancer Immunol. Res. 597):571-581, doi:10.1158 / 2326-6066,CIR-16-0376; Liu et al., 2018, Leukemia 32:520-531).In certain embodiments, the modification of immune effector cells described herein to express cytokines is the same as or follows the methods described in Adachi et al., 2018, Nature Biotechnology, doi:10.1038 / nbt.4086; Liu et al., 2019, J. Immunol., doi:10.4049 / jimmunol.1800033; Krenciute et al., 2017, Cancer Immunol. Res. 597):571-581, doi:10.1158 / 2326-6066,CIR-16-0376; or Liu et al., 2018, Leukemia 32:520-531.

[0268] CAR-expressing immune effector cells In one aspect, provided herein is an immune effector cell comprising any of the chimeric antigen receptors (CARs) described herein.In certain embodiments, provided herein is an immune effector cell transformed with a nucleic acid encoding any of the chimeric antigen receptors (CARs) described herein.In certain embodiments, provided herein is an immune effector cell that expresses any of the chimeric antigen receptors (CARs) described herein.

[0269] Immune effector cells that can be used to carry or express CAR include, but are not limited to, T cells, natural killer (NK) cells, and macrophages. In one embodiment, the immune effector cells are T cells (e.g., cytotoxic T cells). In one embodiment, the immune effector cells are NK cells. In one embodiment, the immune effector cells are macrophages.

[0270] In some aspects, the immune effector cells provided herein are isolated or expanded from peripheral blood, umbilical cord blood, or lymph.

[0271] In some aspects, the immune effector cells provided herein are autologous to the subject to which they are administered (after modification thereof to express a CAR described herein). In certain embodiments, the immune effector cells provided herein are allogeneic to the subject to which they are administered (after modification thereof to express a CAR described herein). When allogeneic immune effector cells are used to prepare CAR-expressing immune effector cells, immune effector cells that reduce the likelihood of graft-versus-host disease in the subject can be selected, or the immune effector cells can be co-administered with one or more immunosuppressants. In some embodiments, the immune effector cells are obtained from a subject, optionally expanded, transformed with a polynucleotide that expresses a CAR described herein, and optionally further expanded.

[0272] In some aspects, immune effector cells are derived from a patient with a disease or condition (e.g., cancer, e.g., AML) and have been genetically modified in vitro to express at least one CAR with specificity for any of the antigens (e.g., neoantigens) described herein. For example, the antigen can be a cancer neoantigen presented by an MHC class I protein (e.g., an antigen comprising a mutant nucleophosmin protein neoepitope complexed with an MHC class I protein, e.g., NPM1c:HLA-A2). In some of these embodiments, immune effector cells genetically modified to express a CAR with specificity for a cancer neoantigen presented by an MHC class I protein (e.g., NPM1c:HLA-A2) are then administered to treat cancer (e.g., NPM1c-positive cancer, e.g., AML) in the patient. In some embodiments, the immune effector cells are stimulated or induced by specific binding of a ligand or antigen to the CAR and perform at least one effector function (e.g., induction of a cytokine) useful for treating the disease or condition in the same patient. In some embodiments, an effector cell is a T cell (e.g., a cytotoxic T cell) that, when in contact with or proximity to a target or target cell (e.g., a cancer cell), exerts its effector function (e.g., a cytotoxic T cell response) on the target cell (see, e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450).

[0273] Stimulation of immune effector cells containing a CAR (e.g., by binding of the extracellular domain of the CAR to a cancer neoantigen) can result in activation of one or more anti-cancer activities of the CAR immune effector cells. For example, in some embodiments, stimulation of the CAR immune effector cells can result in increased cytolytic or helper activity of the CAR immune effector cells, including secretion of cytokines.

[0274] In some embodiments, CAR effector cells (e.g., CAR T cells) comprise a CAR molecule that binds to any antigen described herein (e.g., NPM1c:HLA-A2). In some embodiments, immune effector cells comprising a CAR molecule (e.g., CAR T cells) useful in the methods disclosed herein express a CAR that includes an extracellular domain that binds to an NPM1c neoepitope complexed with (or presented by) an MHC class I protein (e.g., HLA-A2), e.g., NPM1c:HLA-A2. In some embodiments, immune effector cells (e.g., CAR T cells) comprising a CAR molecule useful in the methods disclosed herein express a CAR that includes an NPM1c:HLA-A2 binding domain.

[0275] Prolonged exposure of T cells to their cognate antigens results in the exhaustion of effector functions, allowing the persistence of infected or transformed cells. Recently developed strategies to stimulate or activate host effector functions using agents that induce immune checkpoint blockade have been successful in the treatment of several cancers. Emerging evidence suggests that T cell exhaustion may be a significant obstacle to the sustained long-lived antitumor activity of chimeric antigen receptor-expressing T cells (CAR T cells). The differentiation state of T cells harvested from patients before CAR transduction and the pre-transplant treatments patients receive before reintroduction of CAR T cells (e.g., the addition or elimination of alkylating agents, fludarabine, or total body irradiation) can significantly affect the persistence and cytotoxic potential of CAR T cells. In vitro culture conditions that stimulate (via anti-CD3 / CD28 or stimulator cells) and expand (via cytokines, e.g., IL-2) T cell populations can also alter the differentiation state and effector function of CAR T cells (Ghoneim et al., (2016) Trends in Molecular Medicine 22(12):1000-1011).

[0276] Methods for producing CAR-expressing immune effector cells Provided herein are methods that can be used to generate any of the immune effector cells described herein, including any of the CARs described herein.

[0277] In some embodiments, a subject's immune effector cells (e.g., T cells) are genetically modified with a chimeric antigen receptor (Sadelain et al., Cancer Discov. 3:388-398, 2013). For example, immune effector cells (e.g., T cells) are provided, and a recombinant nucleic acid encoding a chimeric antigen receptor is introduced into the patient-derived immune effector cells (e.g., T cells) to generate CAR cells. In some embodiments, immune effector cells (e.g., T cells) that are not derived from the subject are genetically modified with the chimeric antigen receptor. For example, in some embodiments, the immune effector cells (e.g., T cells) are allogeneic cells engineered to be used as "off-the-shelf" adoptive cell therapy, e.g., universal chimeric antigen receptor T cells (UCART) being developed by Cellectis.

[0278] Any of the nucleic acids encoding a CAR described herein, or an expression vector comprising a nucleic acid encoding a CAR described herein, can be introduced into immune effector cells (e.g., T cells) using a variety of different methods known in the art. Non-limiting examples of methods for introducing nucleic acids into immune effector cells (e.g., T cells) include lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene guns, magnetofection, viral transfection, and nucleofection. Additionally, CRISPR / Cas9 genome editing technology known in the art can be used to introduce CAR nucleic acids into immune effector cells (e.g., T cells) and / or introduce other genetic modifications (e.g., as described below) into immune effector cells (e.g., T cells) to enhance CAR T cell activity (CAR For the use of CRISPR / Cas9 technology in the context of T cells, see, e.g., U.S. Pat. No. 9,890,393; U.S. Pat. No. 9,855,297; U.S. Patent Application Publication No. 2017 / 0175128; U.S. Patent Application Publication No. 2016 / 0184362; U.S. Patent Application Publication No. 2016 / 0272999; WO 2015 / 161276; WO 2014 / 191128; CN 106755088; CN 106591363; CN 106480097; CN 106399375; CN 104894068).

[0279] In some aspects, methods of producing immune effector cells described herein include (i) obtaining cells from peripheral blood, umbilical cord blood, or lymph (e.g., from peripheral blood mononuclear cells (PMBCs)), (ii) optionally purifying the obtained cells, (iii) optionally expanding the cells, (iv) optionally activating the cells (e.g., with an anti-CD3 antibody or antigen-binding fragment thereof and / or an anti-CD28 antibody or antigen-binding fragment thereof), (v) optionally expanding the activated cells, (vi) transducing the cells with an expression vector comprising a CAR described herein, (vii) isolating the cells expressing the CAR, and (viii) optionally expanding the isolated cells.

[0280] In some aspects, the methods for generating immune effector cells described herein include the steps of (i) obtaining pluripotent stem cells (iPSCs), (ii) inducing the iPSCs to become immune effector cells (e.g., NK cells, macrophages, or T cells (e.g., CD8 + (iii) optionally expanding the immune effector cells; (iv) transducing the immune effector cells with an expression vector comprising a CAR described herein; (v) isolating the immune effector cells expressing the CAR; and (vi) optionally expanding the isolated cells.

[0281] composition In one aspect, provided herein is a composition (e.g., a pharmaceutical composition) comprising an antibody or antigen-binding fragment thereof disclosed herein. The antibody or fragment in the pharmaceutical composition may be purified.

[0282] In one aspect, provided herein is a composition (e.g., a pharmaceutical composition) comprising a bispecific molecule (e.g., a bispecific antibody) disclosed herein. The bispecific molecule in the pharmaceutical composition may be purified.

[0283] In one aspect, provided herein is a composition (e.g., a pharmaceutical composition) comprising any of the immune effector cells (e.g., CAR polypeptide-expressing immune effector cells) disclosed herein.

[0284] The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers, including but not limited to excipients and stabilizers, are known in the art (see, for example, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA).

[0285] A pharmaceutical composition may be a sterile composition comprising cells, tethering means (e.g., lipid nanoparticles), and / or proteins or peptides, preferably in a pharmaceutically acceptable carrier (e.g., one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other subjects as contemplated herein). The carrier may be an organic or inorganic component, natural or synthetic, with which the cells, tethering means (e.g., lipid nanoparticles), and / or proteins or peptides are combined to facilitate administration. The components of the pharmaceutical composition are mixed in a manner such that there is no interaction that would substantially impair their desired pharmaceutical efficiency.

[0286] Pharmaceutically acceptable carriers include, but are not limited to, buffers, emulsifying agents, suspending agents, dispersing agents, isotonic agents, wetting agents, chelating agents, sequestering agents, pH buffering agents, solubility enhancers, antibacterial agents, anesthetics, and / or antioxidants.

[0287] Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipient vehicles is considered within the scope of the present disclosure, except insofar as any conventional excipient vehicle may be incompatible with the substance or its derivatives, for example, by producing some undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition. Excipients include, for example, antiadherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, saline, butylhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, sucrose, dextrose, magnesium stearate, malt, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, glycerol, ethanol, polyvinylpyrrolidone, povidone, starch (e.g., pregelatinized starch), propylene, propylparaben, retinyl palmitate, shellac, silica gel, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium stearate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, talc, base cream, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0288] In some embodiments, the pharmaceutical compositions disclosed herein may contain at least one pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts that can be included in the compositions of the present disclosure include, but are not limited to, acid addition salts of basic residues such as amines, alkali or alkaline earth metal salts, mineral or organic acid salts; alkali or organic salts of acidic residues such as carboxylic acids. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethoxybenzoate ... Examples of suitable salts include toluenesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.

[0289] A pharmaceutical composition can be formulated to be suitable for administration to a subject (e.g., a human). The pharmaceutical composition can be formulated for any route of administration.

[0290] Pharmaceutical compositions can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion, when systemic delivery is desired. Such formulations can be prepared as liquid solutions, suspensions, emulsions, or solid forms suitable for solution or suspension prior to injection. Injectable formulations can be provided in unit dosage form, e.g., in ampoules or multidose containers. Pharmaceutical formulations for parenteral use include aqueous solutions of the components. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Alternatively, suspensions of the components can be prepared as oily suspensions. Suitable lipophilic solvents or vehicles include fatty oils, e.g., sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides, or liposomes. For parenteral administration, suitable pharmaceutically acceptable carriers include, but are not limited to, saline or phosphate-buffered saline (PBS), or solutions containing, e.g., polyethylene glycol, polypropylene glycol, or glucose.

[0291] The antibodies or antigen-binding fragments, bispecific molecules, or CAR-expressing immune effector cells described herein can be used or present in the pharmaceutical compositions disclosed herein in a therapeutically effective amount, which can be determined by standard clinical techniques.

[0292] Pharmaceutically acceptable compositions contemplated herein can include, in addition to the antibodies or antigen-binding fragments, bispecific molecules, or CAR-expressing immune effector cells described herein, additional anti-cancer agents (e.g., any one, two, three, or more anti-cancer agents described herein).

[0293] Treatment Methods and Uses In one aspect, the present disclosure provides a method for treating cancer (e.g., inhibiting cancer growth, inhibiting cancer progression) in a subject in need thereof, the method comprising administering to the subject any antibody or antigen-binding fragment described herein, any bispecific molecule described herein, any immune effector cell comprising a CAR polypeptide described herein, or any pharmaceutical composition described herein. In certain embodiments, the present disclosure provides a method for treating NPM1c-positive cancer. As used herein, "NPM1c-positive cancer" refers to a cancer comprising tumor cells with a mutation in the NPM1 gene (e.g., a 4-nt duplication mutation in NPM1), where the mutation in NPM1 results in increased cytoplasmic localization of NPM1 protein compared to cells expressing wild-type NPM1. Methods for measuring gene expression in cancer to determine the presence of specific genetic mutations (e.g., a 4-nt duplication mutation in NPM1) are known in the art and include analysis of malignant tumor samples (e.g., blood, bone marrow, tumor, and / or tissue samples) collected from the subject. In some aspects, methods for detecting small duplications, insertions, or deletions in genes are performed using real-time quantitative polymerase chain reaction (RT-PCR), droplet digital PCR, Sanger sequencing, and next-generation sequencing (e.g., whole-genome sequencing, e.g., whole-exome sequencing). In some aspects, NPM1c-positive cancers are detected to have a mutation in the NPM1 gene (e.g., a 4-base-pair frameshift insertion in exon 12 of the gene, a mutation encoding the C-terminal 11 amino acids in an alternative reading frame, or an NPM1 mutation that results in expression of a protein containing the following C-terminal amino acid sequence: MTDQEAIQDLCLAVEEVSLRK (SEQ ID NO: 57)). In some embodiments, NPM1c-positive cancers include tumor cells with increased cytoplasmic localization of NPM1 protein. Methods for assessing the cellular localization of NPM1 are known in the art, for example, using a labeled anti-NPM1 antibody to assess localization by microscopy or flow cytometry.In some embodiments, tumor cells isolated from NPM1c-positive cancers have increased cytoplasmic localization of NPM1 protein when compared to cells isolated from healthy, non-cancerous tissue samples.

[0294] In some embodiments, the present disclosure provides a method for treating NPM1c-positive cancer (e.g., inhibiting cancer growth or progression) in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment described herein, a bispecific molecule described herein, an immune effector cell comprising a CAR polypeptide described herein, or a pharmaceutical composition described herein.

[0295] In one aspect, the present disclosure provides a method for treating AML (e.g., inhibiting the growth or progression of AML) in a subject in need thereof, the method comprising administering to the subject any antibody or antigen-binding fragment described herein, any bispecific molecule described herein, any immune effector cell comprising a CAR polypeptide described herein, or any pharmaceutical composition described herein. In some embodiments, the patient has a mutation in the nucleophosmin 1 gene. In some embodiments, the AML comprises NPM1c-positive tumor cells or tumor cells expressing a mutation in the nucleophosmin 1 gene. In certain embodiments, the present disclosure provides for the treatment of NPM1c-positive AML.

[0296] In certain embodiments, the antibodies or antigen-binding fragments thereof (e.g., scFvs), bispecific molecules, CAR polypeptides, immune effector cells comprising CAR polypeptides, or pharmaceutical compositions of the present disclosure can be used in the development of targeted immunotherapies for treating cancer. In some embodiments, the cancer is an NPM1c-positive cancer.

[0297] In certain embodiments, the antibodies or antigen-binding fragments thereof (e.g., scFvs), bispecific molecules, CAR polypeptides, immune effector cells comprising CAR polypeptides, or pharmaceutical compositions of the present disclosure can be used to treat AML. In some embodiments, the AML is NPM1c-positive.

[0298] In certain embodiments, an antibody or antigen-binding fragment thereof (e.g., scFv), bispecific molecule, CAR polypeptide, immune effector cell comprising a CAR polypeptide, or pharmaceutical composition of the present disclosure can be used as a cytotoxic agent to kill AML cells. In some embodiments, the AML cells, or a subpopulation thereof, are NPM1c positive.

[0299] In some embodiments, the present disclosure provides a method for treating NPM1c-positive cancer (e.g., AML) in a subject carrying an allele encoding HLA-A2 (i.e., an HLA-A*02:01 allele). In some embodiments, the NPM1c-positive cancer (e.g., AML) comprises tumor cells that express HLA-A2. Methods for determining HLA expression are known in the art and include flow cytometry, immunohistochemistry, and Western blotting using labeled antibodies that recognize HLA-A2. HLA expression can also be determined by RT-PCR and RNA sequencing.

[0300] In certain embodiments, the present disclosure provides treatment of cancer (e.g., an NPM1c-positive cancer, e.g., AML) in a subject in need thereof, wherein the cell surface of cells comprising the cancer presents an NPM1c neoepitope in complex with an MHC class I protein (e.g., HLA-A2), and the treatment comprises administering to the subject any antibody or antigen-binding fragment described herein, any bispecific molecule described herein, any immune effector cell comprising a CAR polypeptide described herein, or any pharmaceutical composition described herein.

[0301] In certain embodiments, the present disclosure provides treatment of cancer (e.g., an NPM1c-positive cancer, e.g., AML) in a subject in need thereof, wherein the cell surface of cells comprising the cancer presents an AIQDLCLAV (SEQ ID NO: 1) neoepitope complexed with an MHC class I protein (e.g., HLA-A2, or a protein encoded by the HLA-A*02 allele group, e.g., a protein encoded by the HLA-A*02:01 allele), and the treatment comprises administering to the subject any antibody or antigen-binding fragment described herein, any bispecific molecule described herein, any immune effector cell comprising a CAR polypeptide described herein, or any pharmaceutical composition described herein.

[0302] In certain embodiments, the present disclosure provides a method for reducing cancer burden or extending survival in a subject with cancer (e.g., the cancer is NPM1c-positive, e.g., the cancer is AML), comprising administering to the subject any antibody or antigen-binding fragment described herein, any bispecific molecule described herein, any immune effector cell comprising a CAR polypeptide described herein, or any pharmaceutical composition described herein. In certain embodiments, the cell surface of the cancer-containing cells presents an NPM1c neoepitope (e.g., SEQ ID NO: 1) complexed with an MHC class I protein (e.g., HLA-A2).

[0303] In certain embodiments, the present disclosure provides for the prevention of cancer in a subject in remission from cancer, comprising administering to the subject any antibody or antigen-binding fragment described herein, any bispecific molecule described herein, any immune effector cell comprising a CAR polypeptide described herein, or any pharmaceutical composition described herein.

[0304] In one embodiment, the cancer is a recurrent cancer. In one embodiment, the cancer is a refractory cancer. In one embodiment, the cancer is an advanced stage cancer. In one embodiment, the cancer is resistant to one or more other therapies (e.g., chemotherapy, radiation therapy, stem cell transplant, or another immunotherapy).

[0305] In certain embodiments, the present disclosure provides for the prevention of AML in a subject in need thereof, comprising administering to the subject any antibody or antigen-binding fragment described herein, any bispecific molecule described herein, any immune effector cell comprising a CAR polypeptide described herein, or any pharmaceutical composition described herein. In one embodiment, the present disclosure provides for the prevention of AML in a subject in remission from AML.

[0306] In certain embodiments, the cancer to be treated is AML. In some embodiments, the cancer is relapsed AML. In some embodiments, the cancer is refractory AML. In some embodiments, the cancer is advanced AML. In one embodiment, the cancer is AML that is resistant to one or more other therapies (e.g., chemotherapy, radiation therapy, stem cell transplantation, or another immunotherapy).

[0307] The effectiveness of any of the treatments described herein can be evaluated by assessing parameters (e.g., tumor burden) before and after treatment (e.g., on the treated subject or on an animal model of the cancer being treated). Any assay known in the art can be used to evaluate the therapeutic effectiveness of the treatments described herein.

[0308] Method of administration The therapies described herein can be administered to a subject by any suitable means, including, but not limited to, parenteral routes of administration. In some embodiments, the compositions are administered parenterally to a patient. Non-limiting examples of suitable routes of parenteral administration include intravenous, intramuscular, intraarterial, subcutaneous, intratumoral, intrathecal, and intraperitoneal administration. In one embodiment, the therapies described herein are administered intravenously. In one embodiment, the therapies described herein are administered intraperitoneally. In one embodiment, the therapies described herein are administered intramuscularly. In one embodiment, the therapies described herein are administered subcutaneously. In certain embodiments, administration is intravenous, intrathecal, intraosseous, or intraspinal. In one embodiment, the therapies described herein are administered intraspinally or intraspinal canal. In one embodiment, the therapies described herein are administered intrathecally. In one embodiment, the therapies described herein are administered intraosseously. In one embodiment, the therapies described herein are administered intramedullary.

[0309] The appropriate dosage will vary depending on the specific cancer to be treated, the age, weight and physical condition of the subject to be treated, the severity of cancer, the route of administration, the duration of treatment, the responsiveness of the subject to be treated, the nature of simultaneous or combined therapy (if any), the specific route of administration, and similar factors that are within the knowledge and expertise of medical professionals.In certain embodiments, the maximum tolerated dose, i.e., the maximum safe dose according to sound medical judgment, should be used.In preferred embodiments, the treatment method should be administered in an effective amount.An effective amount is the amount of the composition administered that is sufficient to provide medically desired results.

[0310] For example, if a subject has a tumor, an effective amount can be an amount that reduces tumor volume or burden (e.g., determined by imaging the tumor).An effective amount can also be assessed by the presence and / or frequency of cancer cells in blood or other body fluids or tissues (e.g., biopsy samples).If a tumor affects the normal function of tissues or organs, an effective amount can be assessed by measuring the normal function of tissues or organs.

[0311] In certain embodiments, the CAR-expressing immune effector cells are about or at least 1 x 10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×100 10 , 5×100 10 , 1×10 11 , or 5 × 10 11 , 1×10 112 , or 5 × 10 12 (or any value or range therebetween).

[0312] Various dosing schedules for the therapeutic methods described herein are contemplated, including single administration or multiple administrations over a period of time. Methods of administration include, but are not limited to, bolus administration and infusion (e.g., continuous or pulse infusion).

[0313] The treatment regimen for use in the methods described herein can include administering therapy twice a week, once a week, once every two weeks, once every three weeks, once a month or every four weeks, once every six weeks, once every two months or every eight weeks, or once every three months or every twelve weeks.In certain embodiments, the subject is administered a single dose of any of the therapies described herein.In certain embodiments, the subject is administered at least two, at least three, at least four, at least five, at least six, at least eight, or at least ten doses of any of the therapies described herein.In certain embodiments, the therapy described herein is administered daily, every other day, or twice a week.In certain embodiments, the therapy described herein is administered for a period of time, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year.

[0314] In some embodiments, a first treatment period (in which the therapy is administered, e.g., once, twice a week, once a week, twice every two weeks, or once a month) is followed by a rest period (e.g., lasting 1 week, 2 weeks, 3 weeks, 1 or 4 weeks, 6 weeks, 2 or 8 weeks, 3 months, 4 months, 5 months, 6 months, or 1 year) in which the antibody is not administered, and then a second treatment period (in which the therapy is administered, e.g., once, twice a week, once a week, twice every two weeks, or once a month). Such first treatment period and such second treatment period can last, for example, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, or 3 months (the first treatment period can be the same as or different from the second treatment period).

[0315] Patient population Subjects treated according to the methods described herein include, but are not limited to, humans and non-human vertebrates. In certain embodiments, subjects treated according to the methods described herein are mammals, such as domestic pets (e.g., dogs, cats, rabbits, ferrets, etc.), livestock or agricultural animals (e.g., cows, pigs, sheep, goats, pigs, chickens, or other poultry), horses (e.g., purebred horses), monkeys, laboratory animals (e.g., mice, rats, rabbits, etc.), and others. Subjects also include fish and other aquatic species. In a preferred embodiment, the subject treated according to the methods described herein is a human. In one embodiment, the present disclosure can be implemented in any subject believed to be likely to benefit from targeted immunotherapy for the treatment of acute myeloid leukemia (AML). In some embodiments, the present disclosure is for use in subjects with NPM1c-positive cancer (e.g., AML).

[0316] In some aspects, the therapeutic methods and uses of the present disclosure can be carried out on any subject with (e.g., diagnosed with) cancer who can benefit (or is likely to benefit) from any of the immunotherapies described herein. A subject with cancer (e.g., an NPM1c-positive cancer, e.g., AML) is a subject who has detectable cancer cells. The present disclosure contemplates the administration of any antibody or antigen-binding fragment thereof (e.g., scFv) described herein, any bispecific molecule described herein, and any immune effector cell expressing a CAR polypeptide described herein to a subject with cancer (e.g., an NPM1c-positive cancer, e.g., AML).

[0317] In some aspects, the therapeutic methods and uses of the present disclosure can be carried out in any subject with a cancer characterized by (e.g., known to have, predicted to have, or detected to have) a mutation in the NPM1 gene (e.g., a four-base-pair frameshift insertion in exon 12 of the gene, a mutation encoding the C-terminal 11 amino acids in an alternative reading frame, or an NPM1 mutation that results in expression of a protein comprising the following C-terminal amino acid sequence: MTDQEAIQDLCLAVEEVSLRK (SEQ ID NO: 57)). In certain embodiments, the therapeutic methods and uses of the present disclosure can be carried out in any subject with a cancer characterized by (e.g., known to express, predicted to express, or detected to express) a mutant NPM1 protein (e.g., an NPM1c mutant protein with cytoplasmic localization, a protein with a mutation in the C-terminal domain, a mutant protein lacking the folded C-terminal domain, a protein comprising the following C-terminal amino acid sequence: MTDQEAIQDLCLAVEEVSLRK (SEQ ID NO: 57), a protein set forth by SEQ ID NO: 56, or NPM1c). Mutated C-terminal sequences of NPM1c are known in the art (see, e.g., van der Lee et al., 2019, J. Clin. Invest. 129(2):774-785, which is incorporated herein by reference in its entirety; see, e.g., Figure 1). In some embodiments, the therapeutic methods and uses of the present disclosure can be carried out in any subject with cancer, wherein the cell surface of cells comprising the cancer presents (e.g., is known to present, expected to present, or detected to present) a mutant nucleophosmin neoepitope (e.g., an NPM1c neoepitope, e.g., AIQDLCLAV (SEQ ID NO: 1)) complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2).In some aspects, the therapeutic methods and uses of the present disclosure can be carried out in any subject with cancer, in which a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2) presents or displays an NPM1c neoepitope (e.g., AIQDLCLAV (SEQ ID NO: 1)) on the cell surface of cells containing the cancer.

[0318] Optionally, cancer cells of a prospective patient to be treated according to the methods described herein are tested for mutations in the NPM1 gene or protein, or tested to determine whether the cell surface of the cancer-containing cells presents an antigen comprising an NPM1c neoepitope (e.g., AIQDLCLAV (SEQ ID NO: 1)) complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2). In some embodiments, if such testing is positive for a mutation in the NPM1 gene or NPM1 protein, or if it is determined that the cancer cells present an antigen comprising an NPM1c neoepitope (e.g., AIQDLCLAV (SEQ ID NO: 1)) complexed with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2) on their cell surface, the patient is treated according to the methods described herein.

[0319] In some aspects, the therapeutic methods and uses of the present disclosure can be carried out in subjects with acute myeloid leukemia (AML). In one particular embodiment, the therapeutic methods and uses of the present disclosure are carried out in subjects diagnosed with AML.

[0320] Tests for diagnosing cancers treated by the methods described herein are known in the art and would be familiar to ordinary medical practitioners. These clinical tests include, but are not limited to, microscopic analysis, culture-dependent tests (such as culture), and nucleic acid detection tests. These include wet mount, stain-enhanced microscopy, immunomicroscopy (e.g., FISH), hybridization microscopy, particle agglutination, enzyme-linked immunosorbent assay, urine screening test, DNA probe hybridization, serological tests, etc. In addition to performing the clinical tests listed above, physicians will generally take a complete medical history and perform a complete physical examination.

[0321] The method for detecting AML includes, but is not limited to, flow cytometry of PBMC for leukemia cells, followed by PCR and sequencing for NPM1c mutation.The clinical method for diagnosing AML is known in the art.The risk factors for developing AML include smoking, chemotherapy, radiotherapy, certain blood diseases and aging.

[0322] In one embodiment, the subject being treated has been diagnosed with early stage cancer (e.g., AML). In one embodiment, the subject being treated has been diagnosed with advanced stage cancer (e.g., AML).

[0323] In some embodiments, the subject being treated is at any stage of AML progression.

[0324] In some aspects, the subject being treated has previously undergone one or more other cancer treatments (e.g., chemotherapy, radiation therapy, or stem cell transplant). In certain embodiments, the subject being treated has previously undergone one or more other cancer treatments (e.g., chemotherapy, radiation therapy, or stem cell transplant), and the subject's cancer has recurred. In certain embodiments, the subject being treated has previously undergone one or more other cancer treatments (e.g., chemotherapy, radiation therapy, or stem cell transplant), and the subject has developed resistance to the one or more other cancer treatments. In certain embodiments, the subject being treated is in remission (e.g., in partial or complete remission of the cancer). In certain embodiments, the subject being treated is refractory to one or more other cancer treatments (e.g., chemotherapy, radiation therapy, or stem cell transplant).

[0325] In other embodiments, it is contemplated herein to treat a subject at risk of developing cancer who can benefit (or is likely to benefit) from any of the immunotherapies described herein according to the therapeutic methods and uses disclosed herein. A subject at risk of developing cancer (e.g., AML) is a subject who has a higher-than-normal probability of developing cancer. These subjects include, for example, subjects with genetic abnormalities that have been demonstrated to be associated with a higher likelihood of developing cancer, subjects with a familial predisposition to cancer, subjects exposed to cancer-causing agents (i.e., carcinogens) such as tobacco, asbestos, or other toxic chemicals, and subjects who have previously been treated for cancer and are in apparent remission. The present disclosure contemplates administering to a subject at risk of developing cancer (e.g., AML) an antibody or antigen-binding fragment thereof (e.g., scFv), a bispecific molecule described herein, and an immune effector cell expressing a CAR polypeptide described herein.

[0326] In one embodiment, the subject to be treated is an adult. In one embodiment, the subject is a human subject 18 years of age or older. In one embodiment, the subject is a human subject 21 years of age or older. In one embodiment, the subject is a human subject 45 years of age or older. In one embodiment, the subject is a human subject 65 years of age or older. In one embodiment, the subject is a human subject under the age of 18. In one embodiment, the sub...

Claims

1. An antibody, or antigen-binding fragment thereof, that specifically binds to an antigen comprising an NPM1c neoepitope complexed with a class I major histocompatibility complex (MHC class I) protein, the antibody, or antigen-binding fragment thereof, (i) a heavy chain variable region (VH) comprising a heavy chain variable region (VH) complementarity determining region (CDR) 1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 are CDRs of a VH having the amino acid sequence of SEQ ID NO: 5; and (ii) a light chain variable region (VL) comprising a light chain variable region (VL) complementarity determining region (CDR) 1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 are CDRs of a VL having the amino acid sequence of SEQ ID NO: 3; An antibody, or an antigen-binding fragment thereof.

2. The antibody, or antigen-binding fragment thereof, of claim 1, which does not bind or does not substantially bind to (a) the MHC class I protein alone and / or (b) a control peptide complexed with the MHC class I protein.

3. The antibody or antigen-binding fragment thereof described in claim 1, wherein the control peptide is an NY-ESO-1 epitope or an influenza virus M1 epitope.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the NPM1c neoepitope comprises an amino acid sequence selected from AIQDLCLAV (SEQ ID NO: 1) or AIQDLCVAV (SEQ ID NO: 71).

5. The antibody, or antigen-binding fragment thereof, of any one of claims 1 to 4, wherein the MHC class I protein is an HLA-A*02 protein and / or is encoded by the HLA-A*02 allele group.

6. (i) a heavy chain variable region (VH) comprising a VH comprising a VH complementarity-determining region (CDR) 1, a VH CDR2, and a VH CDR3, wherein the VH CDR1 has the amino acid sequence GFTFSSYA (SEQ ID NO: 9), the VH CDR2 has the amino acid sequence ISGSGGST (SEQ ID NO: 10), and the VH CDR3 has the amino acid sequence ARLGYPTTTLLPFDY (SEQ ID NO: 11); and / or (ii) a light chain variable region (VL) comprising a VL comprising a complementarity determining region (CDR) 1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 has the amino acid sequence QSISSY (SEQ ID NO: 6), the VL CD2 has the amino acid sequence AAS (SEQ ID NO: 7), and the VL CD3 has the amino acid sequence QQSYSTPLT (SEQ ID NO: 8).

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that is at least 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 5, and / or the VL comprises an amino acid sequence that is at least 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:

3.

8. scFv, The antibody, or antigen-binding fragment thereof, of any one of claims 1 to 7, wherein the scFv comprises a Gly-Ser linker selected from the group consisting of (Gly4Ser) (SEQ ID NO: 58), (Gly4Ser) (SEQ ID NO: 59), (Gly4Ser) (SEQ ID NO: 60), and (Gly4Ser) (SEQ ID NO: 61).

9. The antibody, or antigen-binding fragment thereof, of claim 8, wherein the scFv has the amino acid sequence of SEQ ID NO:

2.

10. 10. The antibody, or antigen-binding fragment thereof, of any one of claims 1 to 9, which is an antibody selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody isotypes.

11. The antibody, or antigen-binding fragment thereof, of any one of claims 1 to 10, wherein the antigen is on the surface of a cancer cell.

12. The antibody, or antigen-binding fragment thereof, of claim 11, wherein the cancer is acute myeloid leukemia (AML).

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, which is a bispecific antibody or antigen-binding fragment thereof that further specifically binds to a second antigen on an immune effector cell, wherein the immune effector cell is a T cell, a natural killer cell, or a macrophage.

14. The antibody, or antigen-binding fragment thereof, of claim 13, wherein the second antigen is CD3, NKp46, CD16A, CD40, CD47, 4-1BB, TGF-β, LAG-3, PD-1, TIM-3, CTLA-4, OX40, NKp30, NKG2A, NKG2D, or DNAM-1.

15. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A chimeric antigen receptor (CAR) polypeptide comprising an intracellular domain, a transmembrane domain, and an extracellular binding domain, wherein the extracellular binding domain comprises the antibody of any one of claims 1 to 9, 13, and 14, or an antigen-binding fragment thereof.

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