Human CXCR5 mRNA

Adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory leukemia and myeloma. Reduced CXCR4 expression correlates with inhibition of NK cell migration to its cognate ligand, stromal-derived factor 1α (SDF-1α), in vitro. NK cells isolated from patients with WHIM syndrome carry a gain-of-function (GOF) mutation in CXCR4 (CXCR4R^334X). Compared with healthy donors, WHIM patient-expanded NK cells were observed to have similar surface levels of CXCR4 but a stronger propensity to home to the bone marrow compartment when adoptively infused into NOD-scid IL2Rgammanull (NSG) mice. Therefore, to enhance the ability of adoptively infused NK cells to home to the bone marrow, in vitro expanded NK cells were genetically engineered to express the naturally occurring GOF CXCR4R^334X receptor variant. Transfection of CXCR4R^334X-encoding mRNA into in vitro expanded NK cells using a clinically applicable approach resulted in sustained increases in cell surface CXCR4 expression without altering NK cell phenotype, cytotoxic function, or reducing NK cell viability. NK cells engineered with CXCR4R^334X showed significantly enhanced chemotaxis to SDF-1α in vitro compared with untransfected and wild-type CXCR4-encoding mRNA-transfected NK cells. Importantly, expression of CXCR4R^334X on expanded NK cells significantly enhanced bone marrow homing after adoptive transfer into NSG mice compared with untransfected NK cell controls. Taken together, these data suggest that upregulating CXCR4R^334X expression on the surface of in vitro expanded NK cells by mRNA transfection is a novel approach to improve homing and target NK cell-based immunotherapy to the bone marrow, the site of hematologic malignancies.

Given the high transfection efficiency and in vitro migration capacity of NK cells treated with CXCR4R^334X mRNA electroporation, the researchers next performed studies to confirm that key features of these NK cell populations, including their phenotype and cytotoxic function, were preserved. As shown in Figure 1, NK cells transfected with CXCR4 mRNA did not alter their activating or inhibitory receptor profiles compared to untransfected control NK cells (Figure 1A). Importantly, NK cell mRNA electroporation did not alter the expression of other surface markers critical for lymphocyte homing and migration across endothelial cells, such as CD62L, integrin α4 (CD49d), and CD44. In addition, mRNA transfection did not affect the ability of NK cells to produce cytokines (IFN-γ and TNF-α) and their cytotoxic function against the gold standard NK cell tumor target K562 (Figure 1B-D). Importantly, NK cells electroporated with CXCR4 mRNA retained high cytotoxic function against leukemia, lymphoma, and multiple myeloma cell lines, as assessed by CD107a degranulation (Figure 1E). Together, these data suggest that mRNA transfection of NK cells is an effective method to transiently introduce novel CXCR4 molecules onto the NK cell surface and that transfection of NK cells expressing CXCR4R^334X can improve NK cell chemotaxis toward SDF-1α without negatively affecting cell viability, phenotype, or function.

Figure 1. NK cells transfected with CXCR4 coding mRNAs have no alterations in NK cell inhibitory and activating receptors and have preserved anti-tumor cytotoxic function. (Levy E, et al., 2019)