# US 11,896,619 B2 · Compositions and methods for immunotherapy of NPM1c-positive cancer

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- Number: US 11,896,619 B2
- Document ID: US11896619B2
- Status: Granted
- Kind: B2
- Application number: 17/144,834
- Filed: 2021-01-08
- Published: 2021-09-16
- Granted: 2024-02-13
- Earliest claimed: 2020-03-10
- Assignees: Massachusetts Institute of Technology
- Applicants: Massachusetts Institute of Technology
- Inventors: Jianzhu Chen; Guozhu Xie
- Web page: https://patentgrep.com/patent/US11896619B2

## CPC

- C07K 16/18: against material from animals or humans
- C07K 16/2818: {against CD28 or CD152}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/42: Cancer antigens
- A61K 40/4211: {CD19 or B4}
- A61P 35/04: specific for metastasis
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/30: from tumour cells
- C07K 16/3061: {Blood cells}
- A61K 2039/505: {comprising antibodies}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/21: from primates, e.g. man
- C07K 2317/32: specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/00: Fusion polypeptide
- C07K 2319/03: containing a transmembrane segment
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## 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) in complex with, or presented by, a class I major histocompatibility complex (MHC class I) protein, or cells expressing such compounds, and their use in methods for treating, or ameliorating one or more symptoms of, cancer.

## Claims

### Claim 1

An antibody, or antigen binding fragment thereof, that specifically binds to an antigen comprising an NPM1c neoepitope in complex with a class I major histocompatibility complex (MHC class I) protein, wherein the antibody, or antigen binding fragment comprises:
(a) a heavy chain variable region (VH), wherein the VH comprises VH complementarity determining region (CDR)1, VH CDR2 and 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
(b) a light chain variable region (VL), wherein the VL comprises VL complementarity determining region (CDR)1, VL CDR2 and VL CDR3, wherein the VL CDR1 has the amino acid sequence QSISSY (SEQ ID NO:6), the VL CDR2 has the amino acid sequence AAS (SEQ ID NO:7), and the VL CDR3 has the amino acid sequence QQSYSTPLT (SEQ ID NO:8).

### Claim 2 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, which does not bind to, or substantially does not bind to: (a) the MHC class I protein alone, and/or (b) a control peptide in complex with the WIC class I protein, wherein the control peptide is an NY-ESO-1 epitope or influenza virus M1 epitope.

### Claim 3 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the NPM1c neoepitope comprises the amino acid sequence selected from: AIQDLCLAV (SEQ ID NO:1).

### Claim 4 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the neoepitope is 7, 8, 9, 10, 11, or 12 amino acid residues in length.

### Claim 5 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the MEW class I protein is an HLA-A*02 protein or is encoded by the HLA-A*02 allele group.

### Claim 6 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the VH comprises an amino acid sequence of SEQ ID NO:5.

### Claim 7 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the VL comprises an amino acid sequence of SEQ ID NO:3.

### Claim 8 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, which is a human antibody or antigen binding fragment, a humanized antibody or antigen binding fragment or a chimeric antibody or antigen binding fragment.

### Claim 9 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the MEW class I protein is encoded by the HLA-A*02:01 allele.

### Claim 10 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO:5 and the VL comprises the amino acid sequence of SEQ ID NO:3.

### Claim 11 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, which 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.

### Claim 12 (depends on 11)

The antibody, or antigen binding fragment thereof, of claim 11, which is an scFv.

### Claim 13 (depends on 12)

The antibody, or antigen binding fragment thereof, of claim 12, wherein the scFv comprises a linker.

### Claim 14 (depends on 13)

The antibody, or antigen binding fragment thereof, of claim 13, wherein the linker is a peptide linker.

### Claim 15 (depends on 14)

The antibody, or antigen binding fragment thereof, of claim 14, wherein the peptide linker is a Gly-Ser.

### Claim 16 (depends on 15)

The antibody, or antigen binding fragment thereof, of claim 15, wherein the Gly-Ser linker is selected from the group consisting of (Gly4Ser) (SEQ ID NO:58), (Gly4Ser)2 (SEQ ID NO:59), (Gly4Ser)3 (SEQ ID NO:60), and (Gly4Ser)4 (SEQ ID NO:61).

### Claim 17 (depends on 15)

The antibody, or antigen binding fragment thereof, of claim 15, wherein the Gly-Ser linker comprises the amino acid sequence SGSSGGSSSG (SEQ ID NO:4).

### Claim 18 (depends on 12)

The antibody, or antigen binding fragment thereof, of claim 12, wherein the scFv comprises the amino acid sequence of SEQ ID NO:2.

### Claim 19 (depends on 18)

The antibody, or antigen binding fragment thereof, of claim 18, wherein the scFv has an amino acid sequence of SEQ ID NO:2.

### Claim 20 (depends on 12)

The antibody, or antigen binding fragment thereof, of claim 12, wherein the scFv is a human scFv.

### Claim 21 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, which is an antibody selected from the group consisting of an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, an IgD, and an IgE antibody isotypes.

### Claim 22 (depends on 21)

The antibody, or antigen binding fragment thereof, of claim 21, wherein the antibody is an IgG1 isotype or IgG4 isotype.

### Claim 23 (depends on 22)

The antibody, or antigen binding fragment thereof, of claim 22, wherein the antibody comprises a wild type IgG1 heavy chain constant region or wild type IgG4 heavy chain constant region.

### Claim 24 (depends on 22)

The antibody, or antigen binding fragment thereof, of claim 22, wherein the antibody comprises a mutant IgG1 heavy chain constant region or mutant IgG4 heavy chain constant region.

### Claim 25 (depends on 24)

The antibody, or antigen binding fragment thereof, of claim 24, wherein the antibody comprises a mutant IgG4 heavy chain constant region, wherein the mutant IgG4 heavy chain constant region comprises any one of the following substitutions: S228P, L235E, L235A, or a combination thereof, according to EU numbering.

### Claim 26 (depends on 25)

The antibody, or antigen binding fragment thereof, of claim 25, wherein the antibody comprises an Fc domain comprising at least one mutation.

### Claim 27 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the antibody or antigen binding fragment thereof is purified.

### Claim 28 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1, wherein the antigen is on the surface of a cancer cell.

### Claim 29 (depends on 28)

The antibody, or antigen binding fragment thereof, of claim 28, wherein the cancer is Acute Myeloid Leukemia (AML).

### Claim 30 (depends on 1)

The antibody, or antigen binding fragment thereof, of claim 1 which is a bispecific antibody, or antigen binding fragment thereof, which further specifically binds to a second antigen on an immune effector cell.

### Claim 31 (depends on 30)

The antibody, or antigen binding fragment thereof, of claim 30, wherein the second antigen is CD3.

### Claim 32 (depends on 31)

The antibody, or antigen binding fragment thereof, of claim 31, wherein the CD3 is a human CD3 expressed on T cells.

### Claim 33 (depends on 30)

The antibody, or antigen binding fragment thereof, of claim 30, wherein the second antigen is NKp46.

### Claim 34 (depends on 33)

The antibody, or antigen binding fragment thereof, of claim 33, wherein the NKp46 is a human NKp46 expressed on NK cells.

### Claim 35 (depends on 30)

The antibody, or antigen binding fragment thereof, of claim 30, wherein the second antigen is CD16A.

### Claim 36 (depends on 35)

The antibody, or antigen binding fragment thereof, of claim 35, wherein the CD16A is a human CD16A expressed on NK-cells.

### Claim 37 (depends on 30)

The antibody, or antigen binding fragment thereof, of claim 30, wherein 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.

### Claim 38 (depends on 1)

An isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding the antibody, or antigen binding fragment thereof, according to claim 1.

### Claim 39 (depends on 38)

The isolated nucleic acid of claim 38, which comprises the nucleotide sequence of SEQ ID NO:12.

### Claim 40 (depends on 38)

An expression vector comprising the nucleic acid of claim 38.

### Claim 41 (depends on 40)

A cell transformed with the expression vector of claim 40.

### Claim 42 (depends on 41)

A method for producing an antibody, or an antigen binding fragment thereof, that specifically binds to an antigen comprising an NPM1c neoepitope in complex with a class I major histocompatibility complex (MHC class I) protein, the method comprising maintaining the cell of claim 41 under conditions permitting expression of the antibody, or antigen binding fragment thereof.

### Claim 43 (depends on 42)

The method of claim 42, further comprising purifying the antibody, or antigen binding fragment thereof.

### Claim 44 (depends on 1)

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, or antigen binding fragment thereof, of claim 1.

### Claim 45 (depends on 44)

The CAR polypeptide of claim 44, wherein
the transmembrane domain comprises the transmembrane domain of CD3-zeta, CD8, CD28, NKG2D, CD16, NKp44 or NKp46.

### Claim 46 (depends on 44)

The CAR polypeptide of claim 44, wherein 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.

### Claim 47 (depends on 44)

The CAR polypeptide of claim 44, wherein the intracellular domain comprises a CD3-zeta signaling domain and a 4-1BB costimulatory domain; wherein the transmembrane domain comprises a CD8 transmembrane domain, and wherein the CAR polypeptide further comprises a CD8 hinge region.

### Claim 48 (depends on 44)

The CAR polypeptide of claim 44, wherein the intracellular domain comprises a CD3-zeta signaling domain comprising the amino acid sequence of SEQ ID NO: 27, and a 4-1BB costimulatory domain comprising the amino acid sequence of SEQ ID NO: 26; wherein 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 of SEQ ID NO: 25; and wherein the CAR polypeptide further comprises a leading sequence comprising the amino acid sequence set forth in SEQ ID NO: 23.

### Claim 49 (depends on 44)

The CAR polypeptide of claim 44, wherein the extracellular binding domain is an scFv comprising the amino acid sequence of SEQ ID NO:24.

### Claim 50 (depends on 44)

The CAR polypeptide of claim 44, which comprises the amino acid sequence set forth in SEQ ID NO: 22.

### Claim 51 (depends on 44)

The CAR polypeptide of claim 44, wherein the CAR polypeptide further comprises a self-cleaving peptide sequence and a cytokine, wherein cleavage of the self-cleaving peptide releases the cytokine.

### Claim 52 (depends on 51)

The CAR polypeptide of claim 51, wherein the cytokine is IL-12, IL-7, IL-13, IL-15, TNF-α, IFN-γ, or CCL19.

### Claim 53 (depends on 44)

An isolated nucleic acid encoding the CAR polypeptide of claim 44.

### Claim 54 (depends on 53)

The isolated nucleic acid of claim 53, which comprises the nucleotide sequence of SEQ ID NO:30.

### Claim 55 (depends on 53)

An expression vector comprising the isolated nucleic acid of claim 53, wherein the expression vector is a viral expression vector or a non-viral expression vector.

### Claim 56 (depends on 55)

The expression vector of claim 55, wherein the expression vector is a viral expression vector, and wherein the viral expression vector is a lentiviral expression vector.

### Claim 57 (depends on 55)

A cell transformed with the expression vector of claim 55.

### Claim 58 (depends on 57)

A method for producing the cell of claim 57, wherein the method comprises:
(i) purifying a cell from peripheral blood mononuclear cells (PBMC),
(ii) optionally, activating the cell 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 cell with the expression vector,
(iv) isolating the cell expressing the CAR polypeptide, and
(v) optionally, expanding the isolated cell.

### Claim 59 (depends on 44)

A cell expressing the CAR polypeptide of claim 44.

### Claim 60 (depends on 59)

The cell of claim 59, wherein the cell is an immune effector cell, wherein expression of the CAR polypeptide targets the immune effector cell to a cancer cell expressing an antigen comprising an NPM1c neoepitope in complex with a class I major histocompatibility complex (MEW class I) protein.

### Claim 61 (depends on 60)

The cell of claim 60, wherein the MHC Class I protein is an HLA-A*02 protein or is encoded by the HLA-A*02 allele group.

### Claim 62 (depends on 60)

The cell of claim 60, wherein the immune effector cell does not substantially target and/or induce killing of a cancer cell expressing wild type NPM1.

### Claim 63 (depends on 60)

The cell of claim 60, wherein the cancer cell is an acute myeloid leukemia (AML) cell.

### Claim 64 (depends on 59)

The cell of claim 59, wherein the cell is a T cell.

### Claim 65 (depends on 64)

The cell of claim 64 wherein the T cell is a human CD8+ T cell.

### Claim 66 (depends on 59)

The cell of claim 59, wherein the cell is a Natural Killer (NK) cell.

### Claim 67 (depends on 59)

The cell of claim 59, wherein the cell is a macrophage.

### Claim 68 (depends on 55, 59)

A method for producing the cell of claim 59, wherein the method comprises:
(i) inducing a pluripotent stem cell (iPSC) to differentiate into an immune effector cell,
(ii) transducing the immune effector cell with the expression vector of claim 55,
(iii) isolating the immune effector cell expressing the CAR polypeptide, and
(iv) optionally, expanding the isolated immune effector cell.

### Claim 69 (depends on 68)

The method of claim 68, wherein the immune effector cell is a NK cell.

### Claim 70 (depends on 68)

The method of claim 68, wherein the immune effector cell is a macrophage.

### Claim 71 (depends on 59)

A pharmaceutical composition, comprising the cell of claim 59, and a pharmaceutically acceptable carrier.

### Claim 72 (depends on 71)

A method of treating acute myeloid leukemia (AML) in a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 71, in an amount sufficient to treat AML.

### Claim 73 (depends on 72)

The method of claim 72, wherein the AML is a relapsed AML or a refractory AML.

### Claim 74 (depends on 1)

A method of treating a cancer in a subject in need thereof, wherein the cell surface of cells comprising the cancer displays an NPM1c neoepitope in complex with a class I major histocompatibility complex (MHC class I) protein, the method comprising administering to the subject the antibody, or antigen binding fragment thereof, of claim 1, in an amount sufficient to treat the cancer.

### Claim 75 (depends on 74)

The method of claim 74, wherein the cancer is acute myeloid leukemia (AML).

### Claim 76 (depends on 74)

The method of claim 74, wherein the method of treating cancer is a method of reducing cancer burden or a method of increasing survival in the subject.

### Claim 77 (depends on 74)

The method of claim 74, wherein the administering is intravenous, intrathecal, intraosseous, or into the spinal cord.

### Claim 78 (depends on 74)

The method of claim 74, wherein the method further comprises administering one or more additional therapeutic agents or procedures.

### Claim 79 (depends on 78)

The method of claim 78, wherein the additional therapeutic agent is an inhibitor of an immune checkpoint molecule; wherein the immune checkpoint molecule is TIM-3, PD-1, PD-L1 or CTLA-4; wherein the inhibitor is an immune checkpoint molecule antibody.

### Claim 80 (depends on 74)

The method of claim 74, wherein the subject is a human.

### Claim 81 (depends on 1)

A kit comprising: (i) the antibody, or antigen binding fragment thereof, of claim 1, (ii) optionally, one or more additional therapeutic agents, and (iii) instructions for use in treating cancer in a subject.

### Claim 82

A pharmaceutical composition comprising a therapeutically effective amount of an antibody, or antigen binding fragment thereof, that specifically binds to a n antigen comprising an NPM1c neoepitope in complex with a class I major histocompatibility complex (MHC class I) protein, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen binding fragment comprises:
(a) a heavy chain variable region (VH), wherein the VH comprises VH complementarity determining region (CDR)1, VH CDR2 and 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
(b) a light chain variable region (VL), wherein the VL comprises VL complementarity determining region (CDR)1, VL CDR2 and VL CDR3, wherein the VL CDR1 has the amino acid sequence QSISSY (SEQ ID NO:6), the VL CDR2 has the amino acid sequence AAS (SEQ ID NO:7), and the VL CDR3 has the amino acid sequence QQSYSTPLT (SEQ ID NO:8).