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EP-4739341-A1 - MODIFIED NK CELL EXPRESSING A CHIMERIC ANTIGEN RECEPTOR

EP4739341A1EP 4739341 A1EP4739341 A1EP 4739341A1EP-4739341-A1

Abstract

The present invention generally relates to the field of cell therapy. In particular, the invention relates to a modified natural killer (NK) cell which is characterized by the inactivation of the endogenous CD45 and CD38 genes, the expression of a chimeric antigen receptor (CAR) which is directed against CD45 and/or CD38, and the inability to proliferate. The modified NK cell of the invention exerts cytotoxicity against malignant cells upon its contacting with a malignant cell. Accordingly, the modified NK cell of the invention can be applied in cell therapy approaches, in particular for the treatment of cancer or immunological diseases. The cell is also particularly useful for stem cell transplantation. In yet another aspect, the invention relates to a method of producing the above NK cell. The method comprises inactivating the endogenous CD45 and CD38 genes of the NK cell, modifying the NK cell to express a CAR directed against CD45 and/or CD38, and irradiating the NK cell such that it loses its ability to proliferate.

Inventors

  • Ayuk, Ayuketang Francis
  • FEHSE, BORIS
  • Harfmann, Maraike
  • GLOW, DAWID
  • SCHROEDER, TANJA

Assignees

  • Universitätsklinikum Hamburg-Eppendorf

Dates

Publication Date
20260513
Application Date
20240705

Claims (15)

  1. 1. Modified natural killer (NK) cell, wherein said cell is characterized by (a) the inactivation of the endogenous CD45 and CD38 genes, (b) the expression of a chimeric antigen receptor (CAR) which is directed against CD45 and/or a CAR which is directed against CD38, (c) the inability to proliferate, wherein said cell exerts cytotoxicity against malignant cells upon its contacting with a malignant cell.
  2. 2. Modified NK cell of claim 1, wherein said NK cell is a human cell.
  3. 3. Modified NK cell of any of claims 1 -2, wherein said NK cell comprises a point mutation in exon 7 of the p53 gene, wherein said point mutation preferably is a C to T at nucleotide 877 in codon 248 of the p53 gene.
  4. 4. Modified NK cell of any of claims 1-3, wherein said NK cell is a modified cell of the cell line KHYG-1, which has been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number ACC 725, or a cell line derived from KHYG-1.
  5. 5. Modified NK cell of any of claims 1-4, wherein said NK cell expresses a CAR which is directed against CD45 and a CAR which is directed against CD38.
  6. 6. Modified NK cell of any of claims 1-5, wherein said NK cell expresses an additional CAR, wherein said additional CAR is directed to a protein selected from the group consisting of B Cell Maturation Antigen (BCMA), Kappa or lambda light chain, Lewis Y, CLL-1, G protein coupled receptor, class C group 5 member D (GPRC5D), Fc receptor-homolog 5 (FcRH5), Human Semaphorin-4A (Sema4A)HLA-Al, HLA-A2,, TCR alpha/beta, TCR gamma/delta, CD3, CD4, CD5, CD7, CD10, CDl la, CD13, CD16, CD19, CD20, CD22, CD23, CD26, CD30, CD33, CD34, CD37, CD38, CD43, CD44v6, CD45, CD56, CD59, CD79a, CD79b, CD90, CD117, CD123, CD133, CD138 and CD319, and wherein said additional CAR is most preferably directed to CD 123 or Sema4A.
  7. 7. Modified NK cell of any of claims 1-6 for use in medicine.
  8. 8. Modified NK cell of any of claims 1-6 for use in a method of treating a cancer disease or an immunological disease in a patient, wherein said cancer disease is preferably selected from the group consisting of leukemia, lymphoma, myeloma, myelodysplastic syndromes, myeloproliferative neoplasms (MPN).
  9. 9. Modified NK cell for use in a method of claim 8, wherein said NK cell is autologous or allogeneic in respect of the patient to be treated.
  10. 10. Method of producing a NK cell which is unable to proliferate and exerts cytotoxicity against target cells, and preferably malignant cells, upon its contacting with the target cells, said method comprising (a) providing an NK cell, (b) inactivating the endogenous CD45 and CD38 genes of the NK cell, (c) modifying the NK cell such that it expresses a CAR directed against CD45 and/or a CAR directed against CD38, and (d) irradiating the NK cell such that it loses its ability to proliferate.
  11. 11. Method of claim 10, wherein irradiating the NK cell is performed at 5-50 Gray.
  12. 12. Method of any of claims 10-11, wherein inactivation step (b) can be performed either before, simultaneous with or after modification step (c), wherein inactivation step (b) is preferably performed before modification step (c), and wherein inactivation step (b) is most preferably performed 12-36 hours before modification step (c).
  13. 13. Method of any of claims 10-12, wherein (i) inactivation step (b) is performed by targeted genome editing, and preferably by using CRISPR/CAS or TALENs; and/or (ii) modification step (c) is performed with a viral or non-viral vector.
  14. 14. Method of any of claims 10-13, wherein the method further comprises step (e) in which the NK cell obtained after performing steps (a)-(c) or (a)-(d) is tested for CD45 and CD45-CAR expression and/or for CD38 and CD38-CAR expression.
  15. 15. Method of any of claims 10-14, wherein the method comprises modifying the NK cell such that it expresses both a CAR directed against CD45 and a CAR directed against

Description

MODIFIED NK CELL EXPRESSING A CHIMERIC ANTIGEN RECEPTOR The present invention generally relates to the field of cell therapy. In particular, the invention relates to a modified natural killer (NK) cell which is characterized by the inactivation of the endogenous CD45 and CD38 genes, the expression of a chimeric antigen receptor (CAR) which is directed against CD45 and/or CD38, and the inability to proliferate. The modified NK cell of the invention exerts cytotoxicity against malignant cells upon its contacting with a malignant cell. Accordingly, the modified NK cell of the invention can be applied in cell therapy approaches, in particular for the treatment of cancer or immunological diseases. The cell is also particularly useful for stem cell transplantation. In yet another aspect, the invention relates to a method of producing the above NK cell. The method comprises inactivating the endogenous CD45 and CD38 genes of the NK cell, modifying the NK cell to express a CAR directed against CD45 and/or CD38, and irradiating the NK cell such that it loses its ability to proliferate. BACKGROUND OF THE INVENTION Chimeric antigen receptor (CAR)-based cell therapy represents a new and very effective therapeutic modality, particularly for malignant hematologic diseases [1,2]. Autologous T cells are most commonly used as effector cells, but there are now also successful approaches using allogeneic NK cells, e.g. derived from umbilical cord blood [3], Previous clinically successful approaches have used CARs against antigens expressed on the target cells, but not on the effector cells [1-3], When CARs were used against antigens that are also expressed on T cells, fratricide (i.e., mutual destruction of CAR T cells) was observed and this could be circumvented e.g., by knockout of the respective antigens in the effector cells [4-6], Such knockout of target antigens on effector cells is only possible if the survival and function of the effector cells are not affected. In CAR-based therapies, the identification of disease-specific target antigens is challenging. Many of the CARs currently investigated in preclinical and clinical studies bind so-called tumor-associated antigens (TAAs), which are expressed not only on malignant but also on healthy cells, which raises the fear of a considerable ("on-target off-tumor") toxicity of the CAR-T cells. For example, in the case of leukemia, CD45, CD33, CD 123 as well as CD38 are being investigated as possible targets for CAR-based therapy. On the other hand, it is known that CD45 is expressed on almost all hematopoietic cells, while CD33 is expressed in the liver [18], CD123 on endothelial cells [19], and CD38 on respiratory epithelial cells and muscle tissue [20], Therefore, when CAR-T cells are used against such antigens, "safety switches" have been suggested to minimize side effects or to provide better and faster treatment. However, these switches are very elaborate and not always reliable. CD45 has attracted particular interest as a possible target, as this protein is not expressed on non-hematopoietic cells in the human body. Since CD45 is, however, expressed on T lymphocytes and NK cells, a knockout is required for CAR-therapy. Given the central role of CD45 in the regulation of effector cell activation, signal transduction, and cytotoxicity [7], a significant loss of function of the resulting cells must be expected upon knockout of CD45. This was confirmed in early work with the RNK-16 cell line (rat), in which wild-type cells, but not CD45- negative mutants, were cytotoxically active [8], In contrast, it was later shown in the mouse model that CD45-negative primary NK cells exhibit defects in activation, signal transduction and cytokine production, whereas cytotoxic activity was preserved in vitro, albeit to a reduced extent [9, 10], While the mouse data indicate residual functionality of CD45-negative primary NK cells, activation and signal transduction in murine and human NK cells are in part different. For example, murine and human NKG2D differ significantly in structure and the signaling pathways used [11], Binding and "crosslinking" with anti-CD45 antibodies was found to inhibit the cytotoxic effect of primary human NK cells, but not primary human T cells, although the exact mechanism is yet unclear. The antibodies could stimulate a cytotoxic-responsive function of CD45, or trigger an inhibitory effect [12-15]. Data on functionality of CD45-negative T cells are lacking, mainly because CD45 /_ mice develop only very few or no T cells [16], Indeed, the role and relevance of CD45 does not only differ considerably between species, but also between T and NK cells [17], The above considerations likewise apply to CD38, a glycoprotein that is present on many of the different types of immune cells in humans. CD38 appears to play an important role in NK cell activation, signal transduction and cytotoxicity [22, 23], which renders it highly questionable whether T cells or NK cells