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EP-4117716-B1 - METHODS FOR GENERATING ENGINEERED MEMORY-LIKE NK CELLS AND COMPOSITIONS THEREOF

EP4117716B1EP 4117716 B1EP4117716 B1EP 4117716B1EP-4117716-B1

Inventors

  • CHEN, JIANZHU
  • RITZ, JEROME
  • ROMEE, Rizwan
  • DONG, Han
  • XIE, Guozhu
  • HAM, James, Dongjoo

Dates

Publication Date
20260506
Application Date
20210310

Claims (15)

  1. An engineered cytokine-induced memory-like human NK cell or a population of said cells, wherein the engineered NK cell or population of said cells expresses 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 in complex with a class I major histocompatibility complex (MHC class I) protein.
  2. The engineered NK cell or population of cells of claim 1, wherein the extracellular binding domain 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 MHC class I protein, optionally wherein the control peptide is an NY-ESO-1 epitope or influenza virus M1 epitope.
  3. The engineered NK cell or population of cells of claim 1 or 2, wherein the NPM1c neoepitope comprises an amino acid sequence selected from: AIQDLCLAV (SEQ ID NO:1) or AIQDLCVAV (SEQ ID NO: 71).
  4. The engineered NK cell or population of cells of claim 1 or 2, wherein 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).
  5. The engineered NK cell or population of cells of any one of claims 1-4, wherein the MHC class I protein is an HLA-A*02 protein and/or is encoded by the HLA-A*02 allele group.
  6. The engineered NK cell or population of cells of any one of claims 1-5, wherein the extracellular domain comprises: (i) a heavy chain variable region (VH) comprising VH complementarity determining region (CDR)1, VH CDR2 and VH CDR3, said VH CDR1, VH CDR2 and VH CDR3 being the CDRs of a VH that has an amino acid sequence of SEQ ID NO: 5, and/or (ii) a light chain variable region (VL) comprising VL complementarity determining region (CDR)1, VL CDR2 and VL CDR3, said VL CDR1, VL CDR2 and VL CDR3 being the CDRs of a VL that has an amino acid sequence of SEQ ID NO: 3.
  7. The engineered NK cell or population of cells of any one of claims 1-6, wherein the extracellular domain comprises a VH comprising VH CDR1, 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),wherein optionally the extracellular domain comprises a VL comprising VL CDR1, VL CDR2 and 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).
  8. The engineered NK cell or population of cells of any one of claims 1-7, wherein the extracellular domain comprises a scFV, wherein optionally the scFV comprises a Gly-Ser linker 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).
  9. The engineered NK cell or population of cells of claim 8, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 2.
  10. The engineered NK cell or population of cells, according to any one of claims 1-9, wherein the antigen is on the surface of a cancer cell and wherein optionally the cancer is Acute Myeloid Leukemia (AML).
  11. The engineered NK cell or population of cells of any one of claims 1-10, wherein the transmembrane domain comprises the transmembrane domain of CD3-zeta, CD8, CD28, DAP12, 2B4, NKG2D, CD16, NKp44 or NKp46 and/or 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.
  12. The engineered NK cell or population of cells of any one of the claims 1-11, 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; 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 set forth in SEQ ID NO: 25; and wherein the extracellular binding domain comprises the antibody, or antigen binding fragment thereof, and a leading sequence comprising the amino acid sequence set forth in SEQ ID NO: 23.
  13. The engineered NK cell or population of cells of any one of the claims 1-12, wherein the intracellular domain further comprises a self-cleaving peptide sequence and a cytokine, wherein cleavage of the self-cleaving peptide releases the cytokine, wherein optionally the cytokine is IL-12, IL-7, IL-13, IL-15, TNF-α, IFN-y, or CCL19.
  14. The engineered NK cell or population of cells of any one of claims 1-13, which (i) produces increased IFNγ in the presence of one or more cytokines and/or tumor targets relative to a control human NK cell or human NK cell line; (ii) has enhanced antibody-dependent cellular cytotoxicity relative to a control human NK cell or human NK cell line; and/or (iii) has enhanced anti-tumor efficacy relative to a control human NK cell or human NK cell line, optionally wherein the control NK cell is a human NK cell activated in the presence of IL-15 alone, or a human NK cell line activated in the presence of IL-15 alone.
  15. The engineered NK cell or population of cells of any one of claims 1-14, which expresses an IL-15 polypeptide, optionally a human IL-15 polypeptide, wherein optionally the IL-15 polypeptide is a secreted IL-15 polypeptide or a membrane bound IL-15 polypeptide.

Description

BACKGROUND Cell-based immunotherapies are in development for 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 explored 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 NK cells are administered to patients, is one of the cancer treatments currently being tested. These approaches involve the use of autologous NK cells taken from patients that are activated/expanded ex vivo and then reinfused to combat tumors, such as metastatic tumors. Strategies that enhance the persistence, in vivo expansion, and effector functions of ACT NK is needed. Chimeric antigen receptor (CAR) T cell therapy has emerged as one of the strategies for the treatment of cancer. Chimeric antigen receptors (CARs) are genetically-engineered, artificial transmembrane receptors that confer a defined specificity for an antigen (e.g., ligand) onto an immune effector cell (e.g., a T cell, natural killer cell or other immune cell), which results in activation of the effector cell upon recognition and binding to the antigen. However, current CARs targeting lineage-restricted or tumor-associated antigens (TAAs) can be accompanied by severe toxicity 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 the major cause of development of tumor resistance to CAR-T therapies (see Srivastava & Riddell, J IMMUNOL 200: 459 (2018)). Accordingly, more effective CAR therapy strategies are needed. SUMMARY OF THE INVENTION The present invention is set out in the claims and provides an engineered cytokine-induced memory-like human NK cell or a population of said cells, wherein the engineered NK cell or population of said cells expresses 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 in complex with a class I major histocompatibility complex (MHC class I) protein. In some aspects, the disclosure provides an engineered cytokine-induced memory-like (ML) human NK cell or a population of said cells, wherein the engineered NK cell or population of said cells expresses 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 in complex with a class I major histocompatibility complex (MHC class I) protein. In some aspects, the engineered NK cell of the disclosure comprises a CAR comprising an extracellular binding domain that 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 MHC class I protein, optionally wherein the control peptide is an NY-ESO-1 epitope or influenza virus M1 epitope. In some aspects, the engineered NK cell of the disclosure comprises a CAR comprising an extracellular binding domain that binds to an antigen comprising an NPM1c neoepitope in complex with MHC class I protein, wherein the NPM1c neoepitope comprises an amino acid sequence X1X2X3X4X5X6X7X8X9, wherein X1 is selected from A, V, L or I, wherein X2 is selected from A, T, S, V, L, I, M or Q, wherein X3 is selected from Q or N, wherein X4 is selected from D or E, wherein X5 is selected from L, I, V, M, A or F, wherein X6 is selected from C, S, or A, wherein X7 is selected from L, I, V, M, A, or F, wherein X8 is selected from A, V, L or I, and wherein X9 is selected from L, I, V, M or A. In some aspects, X1 is selected from A or V, wherein X2 is selected from V, I, or L, wherein X3 is selected from Q or N, wherein X4 is selected from D or E, wherein X5 is selected from L or I, wherein X6 is selected from C or S, wherein X7 is selected from V, L or I, wherein X8 is selected from A or V, and wherein X9 is selected from V, I, or L. In some aspects, X1 is A, wherein X2 is selected from V, I, or L, wherein X3 is Q, wherein X4 is D, wherein X5 is L, wherein X6 is C, wherein X7 is L, wherein X8 is A, and wherein X9 is selected from V, I, or L. In any of the foregoing or related aspects, the NPM1c neoepitope comprises an amino acid sequence selected from: AIQDLCLAV (SEQ ID NO:1) or AIQDLCVAV (SEQ ID NO: 71). In some aspects, 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 I