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JP-7857376-B2 - Quadricistronic system containing homing receptors or cytokines and chimeric antigen receptors for stable gene modification in cell immunotherapy

JP7857376B2JP 7857376 B2JP7857376 B2JP 7857376B2JP-7857376-B2

Inventors

  • ハンス、ジー．クリンゲマン
  • ローラン、エイチ．ボイセル
  • ジョン、エイチ．リー
  • ネイサン、ティー．シューマー

Assignees

  • イミュニティーバイオ、インコーポレイテッド

Dates

Publication Date
20260512
Application Date
20241108
Priority Date
20180801

Claims (15)

  1. These are recombinant modified natural killer (NK)-92 cells that have been stably transfected with nucleic acids . The nucleic acid encodes an anti-differentiation cluster 123 (CD123) chimeric antigen receptor (CAR), an Fc receptor, and a cytokine; The anti-CD123 CAR comprises an intracellular signaling domain derived from FcεRIγ; The recombinant modified cells express the anti-CD123 CAR and the Fc receptor on the cell surface of the recombinant cells. Recombinant modified NK-92 cells.
  2. The recombinant modified NK-92 cell according to claim 1, wherein the Fc receptor has the amino acid sequence of SEQ ID NO: 12.
  3. Recombinant modified NK-92 cells according to claim 2, wherein the amino acid sequence of sequence number 12 has the F158V mutation.
  4. The recombinant modified NK-92 cell according to claim 1, wherein the cytokine has IL-12 activity.
  5. The recombinant modified NK-92 cell according to claim 4, wherein the cytokine is single-stranded IL-12 having the amino acid sequence of SEQ ID NO: 61.
  6. The recombinant modified NK-92 cell according to claim 4, wherein the cytokine is a heterodimer IL-12 having a p35 component having the amino acid sequence of SEQ ID NO: 58 and a p40 component having the amino acid sequence of SEQ ID NO: 60.
  7. The recombinant modified NK-92 cell according to claim 1 , wherein the anti-CD123 CAR comprises a CD8 hinge region and a CD28 transmembrane domain bound to an intracellular signaling domain from FcεRIγ.
  8. The recombinant modified NK-92 cell according to claim 1, wherein the anti-CD123 CAR comprises an intracellular signaling domain derived from FcεRIγ having the amino acid sequence of SEQ ID NO: 31, a CD8 hinge region having the amino acid sequence of SEQ ID NO: 33, and a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO : 35 .
  9. The recombinant modified NK-92 cell according to claim 8, wherein the anti-CD123 CAR comprises, in a single polypeptide chain, an anti-CD123 scFv moiety, followed by a CD8 hinge region having the amino acid sequence of SEQ ID NO: 33, followed by a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 35, followed by an intracellular signaling domain derived from FcεRIγ having the amino acid sequence of SEQ ID NO: 31.
  10. Recombinant modified NK-92 cells that are stably transfected with recombinant nucleic acids and express anti-CD123 chimeric antigen receptor (CAR), Fc receptor, and cytokines from the recombinant nucleic acids, The aforementioned anti-CD123 CAR contains an intracellular signaling domain derived from FcεRIγ, The anti-CD123 CAR and the Fc receptor are expressed on the cell surface of the recombinant modified NK-92 . Recombinant modified NK-92 cells.
  11. The recombinant modified NK-92 cell according to claim 10 , wherein the Fc receptor has the amino acid sequence of SEQ ID NO: 12 and may have the F158V mutation.
  12. The recombinant modified NK-92 cell according to claim 10 , wherein the cytokine is single-stranded IL-12 having the amino acid sequence of SEQ ID NO: 61.
  13. Recombinant modified NK-92 cells according to claim 10 , wherein the cytokine is a heterodimer IL-12 having a p35 component having the amino acid sequence of SEQ ID NO: 58 and a p40 component having the amino acid sequence of SEQ ID NO: 60.
  14. The recombinant modified NK-92 cell according to claim 10, wherein the anti-CD123 CAR comprises an intracellular signaling domain derived from FcεRIγ having the amino acid sequence of SEQ ID NO: 31, a CD8 hinge region having the amino acid sequence of SEQ ID NO: 33, and a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 35 .
  15. The recombinant modified NK-92 cell according to claim 10, wherein the anti-CD123 CAR comprises, in a single polypeptide chain, an anti-CD123 scFv moiety, followed by a CD8 hinge region having the amino acid sequence of SEQ ID NO: 33, followed by a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO: 35, followed by an intracellular signaling domain derived from FcεRIγ having the amino acid sequence of SEQ ID NO: 31 .

Description

This application claims priority to U.S. Provisional Patent Application No. 62/713,264, filed August 1, 2018; U.S. Provisional Patent Application No. 62/713,278, filed August 1, 2018; U.S. Provisional Patent Application No. 62/713,310, filed August 1, 2018; and U.S. Provisional Patent Application No. 62/713,323, filed August 1, 2018. Each of these applications is incorporated herein by reference in its entirety. The contents of the sequence listing ASCII text file named 104077_0007PCT_Seq_listing_rev004_ST25, with a size of 97KB, were created on August 1, 2019, and submitted electronically via EFS-Web with this application, and are incorporated in their entirety by reference. The field of this invention relates to genetically modified cells that utilize cytotoxic activated natural killer cell lines (NK-92) as a basis for improving immunotherapy against cancer and tumors. The background information includes information that may be helpful in understanding the present invention. This does not constitute an admission that any information provided herein is prior art, or is related to the claims thereof, or that any publications specifically or implicitly referenced are prior art. All publications and patent applications herein are incorporated by reference to the same extent as individual publications or patent applications are specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference conflicts with or contradicts a definition of that term provided herein, the definition provided herein shall apply, and the definition in the reference shall not apply. Cancer immunotherapy based on adopted tumor-specific cytotoxic lymphocytes shows promise in treating patients with malignant tumors. Despite this initial success in certain cancers, tumor treatment remains challenging, primarily due to the immunosuppressive nature of the tumor microenvironment. See Swarts et al. "Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy," Cancer Res, vol. 72, pages 2473–2480, 2012. In addition to modified T cells, NK cell-based immunotherapy is being investigated. Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Natural killer (NK) cells generally make up about 10–15% of circulating lymphocytes and bind to and kill target cells, including virus-infected cells and many malignant cells, nonspecifically with respect to antigens and without prior immune sensitization. (Herberman et al., Science 214:24 (1981)). NK-92 is a cytolytic cancer cell line discovered in the blood of subjects with non-Hodgkin lymphoma and subsequently immortalized ex vivo. NK-92® cells are derived from NK cells but lack the main inhibitory receptors exhibited by normal NK cells and retain most of the activating receptors. However, NK-92® cells do not attack normal cells and do not induce unacceptable immune rejection in humans. A common driver of lymph node metastasis is the hypoxic upregulation of CCR7, a chemokine receptor primarily found in naive T cells and dendritic cells. Upregulation of the CCR7 receptor on hematopoietic NK cells has been previously demonstrated to improve NK cell homing to lymph nodes and allow them to follow the same pathway to lymph node compartments, a common route of metastatic diffusion; however, this has not yet been demonstrated in clinically relevant cell lines. Therefore, there remains a need to improve NK cell and NK cell-based therapies, particularly in the homing and regulation of NK cells within the tumor microenvironment. This is a schematic diagram showing the plasmid pNKAT-CCR7-LP3, which contains the CCR7 receptor for insertion into the AAVS1 gene locus of NK-92® cells.This is a schematic diagram showing the plasmid pCRENFAT-CCL21 containing the NFAT-responsive CCL21 gene.This graph shows the expression of phenotypic markers associated with NK-92® cells in wild-type NK-92® cells and modified NK-92® cells expressing CCR7. (Lane 1: aNK (wild-type); Lane 2: Modified NK-92® cells (MA3); Lane 3: Modified NK-92® cells (MB4); Lane 4: Modified NK-92® cells (MB6); Lane 5: Modified NK-92® cells (ME6); Lane 6: Modified NK-92® cells (MH3); A: Isotype (APC); B: CD54 (ICAM-1); C: NKp30; D: NKG2D)This graph shows the cytotoxic activity of modified NK-92® cells expressing CCR7 against K562 cells.This graph shows the cytotoxic activity of modified NK-92® cells expressing CCR7 against HL-60 cells.This graph shows the activation of the NFAT-luciferase reporter gene in NK-92 cells, as demonstrated by its binding to K562 and SUP-B15 cells (when CD19-CAR mRNA was electroporated into NK-92 cells).This graph shows the activation of the NFAT-luciferase reporter gene in NK-92 cells, as demonstrated by its binding to K562 and SUP-B15 cells (when CD19-CAR mRNA was electroporated into NK-92 cells).This graph shows modified NK-92® cells expressing CCR7 (Mi-aNK) that migrated toward chemokines CCL19