Abstract 2896: Combination of cytokines and PM21-particle stimulation results in robust expansion of memory-like Natural Killer cells with enhanced survival
Authors: Oyer Jeremiah L. , Croom-Perez Tayler J. , Robles-Carrillo Liza D. , Dieffenthaller Thomas A. , Hasan Md Faqrul , Gitto Sarah B. , Mucha Joanna M. , Altomare Deborah A. , Igarashi Robert Y. , Copik Alicja J. 
Published: June 2023

Abstract
In this study we propose to combine feeder cell-free PM21 particle activation of Natural Killer (NK) cells with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce highly proliferative, persistent memory-like NK cells with great therapeutic potential. Development of NK cell therapeutics has risen to prominence in recent years for use as potential anti-cancer therapy. For clinical applications, NK cells are typically either ex vivo activated with cytokines or expanded with feeder cells or by feeder cell-free methods. One such method for expansion uses plasma membrane particles containing surface IL-21 and 41BBL (PM21) that allows for feeder cell-free expansion of highly cytotoxic NK cells. Activation of NK cells with IL-12, IL-15, and IL-18 has been shown to induce memory-like properties in NK cells with enhanced proliferative potential and persistence. In this study, use of cytokines with PM21 particles for NK cell expansion (CAP-NK cells) led to an increase from an average 2,400-fold expansion with PM21-particles alone (PM21-NK cells) to 13,600-fold in 14 days (p&lt;0.01) when grown in flasks. Using G-Rex® Well Plates, a scalable clinically relevant platform, CAP-NK cells could be expanded an average 33-fold in just 7 days compared to 6-fold for PM21-NK cells (p&lt;0.005). CAP NK cells had enhanced proliferation, decreased doubling time, and an increase in the percent of CD25+41BB+ NK cells (14% of PM21-NK cells vs 82% of CAP-NK cells; p&lt;0.0001). CAP-NK cells also demonstrated higher levels of basal and compensatory glycolysis and increased maximal respiration compared to PM21-NK cells. CAP-NK cells demonstrated enhanced effector functions; they were more cytotoxic against ovarian (SKOV-3) and lung (A549) cancer cells compared to PM21-NK cells (SKOV-3: 58% vs 42%, p=0.0002; A549: 77% vs 65% at 24 h at 1:1 E:T, p=0.001) and had increased IFNγ production after stimulation with cytokines or K562 cells. Importantly, CAP-NK cells also demonstrated memory-like properties. After expansion for 14 days, NK cells were cold-washed and rested for 7 days with 1 ng/mL IL-15. Rested CAP-NK cells had increased IFNγ production when re-stimulated with IL-12, IL-15 and IL-18 (MFI 5353 vs 3679; p&lt;0.01) and higher potency against K562 (EC50 0.58 vs 1.17; p&lt;0.05) as compared to rested PM21-NK cells. Finally, cryopreserved CAP-NK cells showed improved persistence 21 days after i.p. injection with no exogenous cytokine support in NSG mice. Additionally, CAP-NK cells could home to and control tumor growth in an orthotopic K562 model. Taken together, the CAP-NK method results in highly proliferative, highly cytotoxic memory-like NK cells amenable to clinical use.
Citation Format: Jeremiah L. Oyer, Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Thomas A. Dieffenthaller, Md Faqrul Hasan, Sarah B. Gitto, Joanna M. Mucha, Deborah A. Altomare, Robert Y. Igarashi, Alicja J. Copik. Combination of cytokines and PM21-particle stimulation results in robust expansion of memory-like Natural Killer cells with enhanced survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2896.