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Abstract
Background Natural killer (NK) cells play a crucial role in immunosurveillance and antitumor immunity via detection and elimination of virally-infected or neoplastic cells.1 2 With a low risk of graft-versus-host disease following allogenic adoptive transfer and potent killing capabilities, NK cells have unique potentials for cell-based therapies as shown in recent proof-of-concept clinical trials, particularly via the introduction of targeted chimeric antigen receptors (CAR).3 4 Efforts to maximize therapeutic potential focus on genetic modifications to increase NK cell killing potency, improve persistence, and enhance specificity. While VSV-G pseudotyped lentiviral vectors have been extensively used in engineering T cells and hematopoietic stem/progenitor cells (HSPC) for clinical applications, they have been limited by very low transduction efficiency of NK cells. Baboon endogenous virus envelope (BaEV) lentiviruses offer an alternative but have limitations in terms of titer and clinical development. Improved methods for efficient modification of NK cells are crucial for advancing CAR-NK cell-based therapies, especially in pre-clinical models such as the rhesus macaque (RM) for testing safety, persistence and efficacy.
Methods Towards our larger goal of investigating the clonal dynamics and the safety, persistence and efficacy of IL15-armored CAR-NK cells compared to unarmored ones, we inserted an antiCD20-CAR with or without the cDNA for soluble RM IL15 into lentiviral backbones and generated VSV-G pseudotyped LV vectors. Transduction efficiency of RM blood NK cells was optimized using 100µg/ml Poloxamer 407 (P407), a non-ionic amphiphilic molecule used for enhancing lentiviral transduction of RM HSPC.5 RM CD3negNKG2Apos NK cells were expanded ex vivo with 500IU/ml rhIL2 and irradiated universal antigen-presenting (uAPC) feeders6 for 5 days prior to LV transduction, followed by nine days additional culture (figure 1A). CAR-NK cells were evaluated in vitro and in immunodeficient mouse xenografts.
Results Efficient lentiviral transduction (40–60%) of RM NK cells was achieved at MOI 20–50 (figure 1B-C). Armored IL-15-secreting CAR-NK cells demonstrated superior cytotoxicity against CD20pos tumor cells in vitro compared to non-armored cells (figure 1D). In xenografts, IL15-armored CAR-NK cells exhibited enhanced persistence, proliferation and reduced tumor burden compared to unarmored CAR-NK cells (figure 1E-G).
Conclusions P407 significantly improved the transduction efficiency of RM NL cells with VSV-G pseudotyped LV vectors. IL15-armored CAR-NK cells showed enhanced persistence, proliferation, and superior therapeutic efficacy in vivo. Ongoing work involves adoptive transfer of IL15-armored and unarmored CAR-CD20 NK cells clonally marked with genetic barcodes into autologous RM, with tracking of their function, persistence, and clonal dynamics.
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Ethics Approval Animal study was approved by NHLBI Animal Care and Use Committee.
A) Schematic representation of experimental design. Rhesus macaque peripheral blood derived CD3negNKG2Apos NK cells were isolated by magnetic cell sorting, and NK cells were expanded and activated ex vivo for 5 days. Activated NK cells were transduced with VSV-G enveloped lentivirus in NK growth media supplemented with 100μg/ml P407. Following second stimulation with irradiated universal antigen presenting cells (uAPC), NK cells were further expanded followed with in vitro and in vivo assays. B) Chimeric antigen receptor (CAR) designs of IL15 secreting or non-secreting lentiviral vectors with or without highly diverse barcode sequence with different library identifications. C) NK transduction efficiency, and transgene expression levels over time compared to untransduced NK (UT NK) cells. D) The cytotoxicity of NK cells against K562 and Raji at 1:1 ratio was measured 4 hours after co-culturing on day 14 post-transduction. E) Mouse CD45neg,nonhuman primate (NHP) CD45pos, NHP NKG2Apos NK cell percentages found by flow in liver over time. D) Proliferation marker ki67 positive NK cell percentages in liver on day 4 post infusion. G) In vivo Imagining System (IVIS) images of firefly-luciferase activity of Raji cells over time. Line graph represents the total flux (p/s) over time in all three experimental groups. Column means were compared with 2way ANOVA for statistical analysis
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