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Chemokine responsive activated natural killer cells with secondary homing activation for verified targets

A technology of chemokine receptors and cells, applied in the direction of chemokines, cytokines/lymphokines/interferon receptors, animal cells, etc.

Pending Publication Date: 2021-03-16
IMMUNITYBIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Upregulation of the CCR7 receptor on blood NK cells has been demonstrated previously to improve NK cell homing to the lymph node, allowing them to follow the same pathway to the lymph node compartment that is a common pathway for metastatic spread, but has not yet been demonstrated in clinically relevant cell lines proven in

Method used

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  • Chemokine responsive activated natural killer cells with secondary homing activation for verified targets
  • Chemokine responsive activated natural killer cells with secondary homing activation for verified targets
  • Chemokine responsive activated natural killer cells with secondary homing activation for verified targets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1. Modified NK cell lines expressing CCR7

[0071] The pNKAT-CCR7-LP3 plasmid ( figure 1 ), followed by gel purification of the linearized plasmid and subsequent electroporation of the linearized plasmid on NK-92 cells using the NEON transfection system (Thermo Fisher Scientific, Waltham, MA) to produce modified NK-92 cells. After 1 week of puromycin selection, the resulting polyclonal populations were tested for CCR7 expression and tested by expression in X-Vivo supplemented with 5% human serum and IL-2. TM Monoclonal cell lines were obtained by serial dilution in culture medium (Lonza, Basel, Switzerland). Improved selection was observed with the addition of 25% conditioned media (sterile filtered supernatant from polyclonal populations). Modified NK-92 cells contain the EF1α promoter all wrapped in homology arms targeting the AAVS1 locus, the CCR7 gene with a poly-A tail, and the loxP-flanked puromycin resistance gene driven by the ubiquitin promoter ( SEQ...

Embodiment 2

[0078] Example 2. Generation of NFAT-responsive constructs for controlled expression of CCL21

[0079] To identify NFAT-responsive elements, a cell line stably expressing an NFAT-based luciferase expression cassette (NR2.2) was generated by electroporation of the linearized construct into Nk-92 cells containing the terminator region, followed by three NFAT-responsive elements (SEQ ID NO:4) and a minimal promoter (SEQ ID NO:5), which thus drive the production of firefly luciferase in the presence of activated NAFT. Subpopulations of these cells were also subsequently electroporated with mRNA containing anti-CD19 CAR (an antigen present on Sup-B15 cells that is otherwise resistant to killing by NK-92 cells). These cells are indicated as ENR2.2 in the left panel. Cells were then plated in triplicate in the absence or presence of target cells and incubated for periods of 2.5 hours to 24 hours. At the end of the incubation period, step 1 reagents from the Promega DualGlo system w...

Embodiment 3

[0080] Example 3. Modified NK cells expressing CCR7 and CCL21.

[0081] The pCRENFAT-CCL21 plasmid was incorporated into CCR7-containing NK-92 cells in a recombinase-mediated cassette exchange using the LoxP site embedded in the pNFAT-CCR7-LP3 construct. Following electroporation of a circular plasmid (pCRENFAT-CCL21), Cre recombinase was transiently expressed, mediating the exchange of the old selection cassette by the new LoxP flanking cassette. Blasticidin selection was used to facilitate the incorporation of new cassettes, and monoclonal cell lines were subcloned from the resulting population in the same manner as previously described in Example 1 to obtain modified NK- 92 cells.

[0082] To evaluate modified NK-92 cells expressing CCR7 and CCL21, unstained modified NK-92 cells were co-cultured with cells known to cause NFAT activation (K562 or other cell lines) in the lower well of a Boyden chamber, and stained The modified NK-92 cells were placed in the upper chamber. ...

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Abstract

Provided herein are modified NK-92 cells comprising a nucleic acid encoding C-C chemokine receptor type 7 (CCR7) operably linked to a promoter. Optionally, the cells further comprise a nucleic acid encoding C-C motif ligand 21 (CCL21), a nucleic acid encoding C-C motif ligand 19 (CCL19) or a combination thereof. Also provided are compositions and kits comprising the modified NK-92 cells. Providedare methods of making the modified cells and methods of treating cancer using the cells.

Description

Background technique [0001] Cancer immunotherapy based on adoptive transfer of tumor-specific cytotoxic lymphocytes is promising for the treatment of patients with malignant tumors. Despite the early success of this therapy in some cancers, the treatment of tumors remains a challenge, mainly due to the immunosuppressive nature of the tumor microenvironment. In addition to modified T cells, NK cell-based immunotherapies are being explored. NK-92 is a cytolytic cancer cell line that was found in the blood of subjects with non-Hodgkin's lymphoma and subsequently immortalized ex vivo. NK-92 cells are derived from NK cells but lack the major inhibitory receptors displayed by normal NK cells while retaining most of the activating receptors. However, NK-92 cells do not attack normal cells and do not cause unacceptable immune rejection in humans. [0002] A common driver of lymph node metastasis is hypoxia-driven upregulation of CCR7, a chemokine receptor predominantly present on n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/52A61K35/17C07K14/715A61K39/00
CPCC07K14/523C07K14/7158A61K35/17A61K39/001121A61K2039/5156A61K2039/5158A61K2039/804A61P35/02C12N5/0646A61P35/00C07K14/521
Inventor N·邵默L·博伊赛尔H·克林格曼
Owner IMMUNITYBIO INC
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