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CN-122029272-A - Optimized IL-15 constructs

CN122029272ACN 122029272 ACN122029272 ACN 122029272ACN-122029272-A

Abstract

Immune effector cells, such as Natural Killer (NK) cells and T cells, are engineered to enhance their cytotoxicity, and the cells and compositions thereof have uses in the treatment of cancer. Engineered immune effector cells and cell lines are prepared by gene engineering to knock down expression of CISH genes, CD38 genes, and/or TGFBR2 genes, and knock in expression of genes encoding IL-15.

Inventors

  • Bruce. MacCready
  • Subassi Saka
  • Tiziano Because of Jenere

Assignees

  • 昂克医疗有限公司

Dates

Publication Date
20260512
Application Date
20241004
Priority Date
20231005

Claims (20)

  1. 1. A human immune effector cell engineered to have reduced CISH expression and to express IL-15, wherein the IL-15 expression is driven by a reduced/weak promoter.
  2. 2. The human immune effector cell of claim 1, wherein the human immune effector cell is an NK cell.
  3. 3. The human immune effector cell of any one of the preceding claims, wherein the reduced/weak promoter results in at least a 25% reduction in induction of IL-15 expression compared to the EF-1 a promoter.
  4. 4. The human immune effector cell of claim 3, wherein the reduced/weak promoter results in at least a 50% reduction in induction of IL-15 expression compared to the EF-1 a promoter.
  5. 5. The human immune effector cell of any one of the preceding claims, wherein the attenuated/weak promoter is selected from MND, UBC and PGK.
  6. 6. The human immune effector cell of any one of the preceding claims, wherein the attenuated/weak promoter is MND.
  7. 7. The human immune effector cell of any one of the preceding claims, wherein the attenuated/weak promoter is MND.
  8. 8. The human immune effector cell of any one of the preceding claims, wherein CISH expression is reduced by at least 50% compared to an identical human immune effector cell that has not been engineered.
  9. 9. The human immune effector cell of any one of the preceding claims, wherein CISH expression is knocked out.
  10. 10. The human immune effector cell of any one of the preceding claims, wherein the IL-15 is expressed at the CISH locus, e.g., within exon 4, thereby knocking out expression of CISH.
  11. 11. The human immune effector cell of any one of the preceding claims, further expressing a Chimeric Antigen Receptor (CAR) that binds, for example, CD38, HER2, VEGF, epCAM, EGFR, BCMA, PD-L1, CD138, CD229, MUC-1, CLL-1, SLAMF7, CD96, CD19, and/or CD47.
  12. 12. The human immune effector cell of any one of the preceding claims, wherein the human immune effector cell is further engineered to have reduced expression of CD38, e.g., wherein CD38 expression is knocked out.
  13. 13. The human immune effector cell of any one of the preceding claims, comprising a bicistronic or polycistronic sequence that expresses a gene encoding IL-15 and a gene encoding a second transgene, e.g., a CAR of claim 10.
  14. 14. The human immune effector cell of claim 13, wherein in the bicistronic or polycistronic sequence, the gene encoding IL-15 is located upstream of the gene encoding a second transgene, such as the CAR of claim 10.
  15. 15. The human NK cell or NK cell line of any of the preceding claims, wherein the cell is engineered to have: a) Knock-out CISH expression, and B) A knocked-in gene encoding IL-15, wherein expression of IL-15 is driven by a promoter that results in at least a 50% reduction in induction of IL-15 expression compared to when IL-15 expression is driven by an EF-1 alpha promoter.
  16. 16. The human NK cell or NK cell line of any of the preceding claims, wherein the cell is engineered to have: a) Knock-out CISH and CD38 expression, and B) Genes encoding IL-15 and CD38 CAR knocked in to CISH loci; wherein the IL-15 and CD38 CAR are driven by a attenuated/weak MND promoter; And wherein the IL-15 and CD38 CAR are expressed from a bicistronic vector and the IL-15 coding sequence is located upstream of the CD38 CAR coding sequence.
  17. 17. The human NK cell or NK cell line of claim 16, wherein the genes encoding IL-15 and CD38 CAR are knocked in exon 4 of the CISH gene.
  18. 18. A human NK cell or NK cell line comprising a bicistronic vector expressing IL-15 and a CAR, wherein the IL-15 transgene is located upstream of the CAR transgene.
  19. 19. The human immune effector cell of any one of the preceding claims, wherein the IL-15 is a soluble secreted form of IL-15 (scIL-15).
  20. 20. The human immune effector cell of any one of the preceding claims, for use in the treatment of cancer, such as hematologic cancer or solid cancer.

Description

Optimized IL-15 constructs Introduction to the invention The present invention relates to engineering immune effector cells (e.g., natural Killer (NK) cells or T cells) and cell lines thereof to produce cells having a more potent cytotoxic phenotype. Furthermore, the invention relates to methods of preparing engineered immune effector cells and cell lines, compositions containing said cells and cell lines, and the use of said cells, cell lines and compositions in the treatment of cancer. Background Typically, immune cells require that the target cell presents antigen via the Major Histocompatibility Complex (MHC) before triggering an immune response that leads to death of the target cell. This allows cancer cells that do not present MHC class I to evade most immune responses. However, NK cells are able to recognize cancer cells in the absence of MHC class I expression. Thus, they play a key role in the body's defense against cancer. On the other hand, in some cases, cancer cells exhibit the ability to attenuate the cytotoxic activity of immune effector cells by expressing ligands that bind to inhibitory receptors on the immune effector cell membrane. Resistance to cancer treatment may involve a balance between these factors and others. In this context, cytotoxicity refers to the ability of immune effector cells (e.g., NK cells or T cells) to induce cancer cell death, e.g., by releasing a cytolytic compound, or by binding to a receptor on the cancer cell membrane and inducing apoptosis of the cancer cell. Cytotoxicity is affected not only by signals that induce release of cytolytic compounds, but also by signals that inhibit their release. Thus, an increase in cytotoxicity will result in a more efficient killing of cancer cells, while the likelihood that cancer cells impair the cytotoxic activity of immune effector cells is reduced, as described above. Cytokine-induced expression of SH2-containing proteins (CIS) is induced by certain growth cytokines and is a key negative regulator of interleukin-15 (IL-15) signaling in immune effector cells such as natural killer cells (NK cells). CIS is encoded by CISH gene. When maintained at low cytokine concentrations, expression of CIS is associated with limited cell expansion and reduced cytotoxic activity against a variety of cancer cell lines. Therefore, it has recently been reported that CISH gene knockout CIS expression in NK cells is useful for cytotoxicity of NK cells against cancer cells (Zhu et al ,2020"Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity" Cell：, volume 27 (2): pages 224-237). Tgfβ receptor (TGFBR) signaling in immune effector cells is known to have immunosuppressive effects and to affect immune effector cell function. TGF-beta alters the expression of effector molecules, activating receptors and chemokine receptors, affects metabolism, and induces the phenotypic conversion of NK cells to less cytotoxic ILC-1 (regions et al 2020"NK Cell Function Regulation by TGF-beta-Induced EPIGENETIC MECHANISMS" Front immunol. 11:311). Immune effector cells (e.g., NK cells or T cells) express IL-15 receptors and thus respond to IL-15 in the cancer microenvironment, IL-15 is typically expressed in soluble and membrane-bound form by other cells of the immune system. Although there is a theoretical interaction between CISH expression and IL-15 signaling, it is not known whether any interaction can be exploited to provide new and improved cancer therapies. There is a need for alternative and preferably improved cell-based therapies, particularly therapies directed against cancer cells, with improved cytotoxicity and persistence. It is an object of the present invention to provide immune effector cells and cell lines that target cancer cells and have a more cytotoxic and/or persistent phenotype. It is a further object to provide methods of preparing engineered immune effector cells and cell lines, compositions containing said cells or cell lines, and their use in therapy, in particular in the treatment of cancer. More specific embodiments are directed to providing treatment for identified cancers. Particular embodiments are directed to combinations of two or more alterations to immune effector cells and cell lines to enhance cytotoxicity and persistence of the altered cells. Disclosure of Invention The present invention provides engineered immune effector cells, such as NK cells or T cells, having a more potent cytotoxic phenotype, and methods of making the cells and cell lines. In addition, the invention provides compositions of immune effector cells (e.g., engineered NK cells or T cells) and cell lines thereof, and uses of the cells/compositions for the treatment of cancer. As described above, cytotoxicity in the present invention means killing cancer cells. The present invention provides immune effector cells (e.g., NK cells or T cells) and cell lines thereof that exhibit (e.g