RNA-Seq Analysis Reveals CCR5 as a Key Target for CRISPR Gene Editing to Regulate In Vivo NK Cell Trafficking

Abstract

A growing number of natural killer (NK) cell-based immunotherapy trials utilize ex vivo expansion to grow and activate allogenic and autologous NK cells prior to administration to patients with malignancies. Recent data in both murine and macaque models have shown that adoptively infused ex vivo expanded NK cells have extensive trafficking into liver tissue, with relatively low levels of homing to other sites where tumors often reside, such as the bone marrow or lymph nodes. Here, we evaluated gene and surface expression of molecules involved in cellular chemotaxis in freshly isolated human NK cells compared with NK cells expanded ex vivo using two different feeder cells lines: Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) or K562 cells with membrane-bound (mb) 4-1BB ligand and interleukin (IL)-21. Expanded NK cells had altered expression in a number of genes that encode chemotactic ligands and chemotactic receptors that impact chemoattraction and chemotaxis. Most notably, we observed drastic downregulation of C-X-C chemokine receptor type 4 (CXCR4) and upregulation of C-C chemokine receptor type 5 (CCR5) transcription and phenotypic expression. clustered regularly interspaced short palindromic repeats (CRISPR) gene editing of CCR5 in expanded NK cells reduced cell trafficking into liver tissue and increased NK cell presence in the circulation following infusion into immunodeficient mice. The findings reported here show that ex vivo expansion alters multiple factors that govern NK cell homing and define a novel approach using CRISPR gene editing that reduces sequestration of NK cells by the liver.

iPSC-Derived Natural Killer Cell Therapies - Expansion and Targeting

“…A detailed transcriptomic analysis demonstrated ex vivo expanded NK cells had drastic differences in expression pattern of chemotactic receptors and ligands, including a significant downregulation of CXCR4 and consequent upregulation of CCR5. The study further observed knocking out CCR5 resulted in reduced NK cell trafficking into liver and corresponding increase in NK cell presence in the blood circulation in immunodeficient mice post-infusion (105). PB-NK cells transfected with CCR7 had increased towards CCL19, a lymph node-associated chemokine (112).…”

“…Efficient tumor infiltration and homing of NK cells is vital for effective anti-tumor activity. However, cells within the TME secrete chemokines such as C-X-C motif chemokine ligand 8 (CXCL8) or C-C motif chemokine ligand 2 (CCL2) that suppress the activity of intratumoral NK cells (105). High concentrations of adenosine in primary and metastatic TME, specifically myeloid cell adenosine A2A receptors (A2ARs) have a myelosuppressive effect that leads to suppression of NK cell anti-tumor activity (106).…”

Improved Activity against Acute Myeloid Leukemia with Chimeric Antigen Receptor (CAR)-NK-92 Cells Designed to Target CD123

“…It is possible that the cells did transit to the bone marrow, but did not persist long enough to be detected at the time of sacrifice or to eliminate AML cells from the bone marrow. Results from others indicate that homing of expanded NK cells into the bone marrow may be inhibited due to increased C-C chemokine receptor type 5 (CCR5) expression and decreased C-X-C chemokine receptor type 4 (CXCR4) expression [ 30 ]. The same group showed improved bone marrow homing of NK cells in NSG mice via transfection with CXCR4 R334X , a gain-of-function variant [ 31 ].…”

Natural killer cell homing and trafficking in tissues and tumors: from biology to application

“… 99 Knocking out CCR5 from expanded NK cells (using CRISPR/Cas9 technology) reduces trafficking into liver tissue but increases the number of NK cells in the circulation and lung following adoptive transfer into immunodeficient mice. 100 Human Eomes hi NK cells show high liver residency, whereas circulating Eomes low NK cells can upregulate Eomes expression if stimulated by cytokines such as IL-15 and transforming growth factor-β (TGF-β) released during inflammation. Upregulated Eomes is believed to increase the expression of CCR5 on NK cells.…”