Abstract P20: Anti-CD123 CAR NK cells and cam161533 Trispecific Killer Engager have synergistic activity against Acute Myeloid Leukemia
Authors: Mendelowitz Alyssa S. , Chu Yaya , Felices Martin , Miller Jeffrey , Forman Stephen J. , Behbehani Gregory , Bonifant Challice , Lee Dean A. , Cairo Mitchell 
Published: March 2024

Abstract
Background Despite advances in allogeneic stem cell transplantation (alloSCT) in both children and adults with acute myeloid leukemia (AML), disease outcomes remain dismal in specific subgroups with estimated overall survival of ≤25%, representing a major patient outcome gap (Cairo, Clin Adv Hematol Oncol. 2017). NK cells are innate immune cells with potent alloreactive anti-AML effects (Dunbar, Haematologica. 2008; Ruggeri et al Science. 2002). Low numbers of circulating NK cells, lack of AML targeting and short persistence have hampered the effectiveness of adoptive NK cell therapy in AML. We have successfully expanded NK cells ex vivo utilizing genetically-engineered feeder cells (Denman/Lee, PLoS One. 2012). To enhance tumor specificity and NK activity we have successfully developed several chimeric antigen receptor (CAR) NK cells by mRNA electroporation. CD123 is expressed on over 80% of AML cells, most importantly on leukemic stem cells, and is considered an ideal immunotherapy target (Jordan, Leukemia, 2000). CD33, another ideal target, is detected on blasts of 85–90% of patients with AML. The cam161533 Tri-specific Killer Engager (TriKE) consists of a camelid agonistic binder to CD16 on NK cells to mediate ADCC, an anti-CD33 binding domain to AML and IL-15 costimulation between the binding domains (Felices/Miller, Cancer Immunol Res. 2020). Objectives To investigate the combinatorial therapeutic effects of anti-CD123 CAR NK with cam161533 TriKE against AML. Methods PBNK cells were ex vivo expanded with lethally irradiated K562-mbIL21-41BBL (Chu/Cairo, JITC, 2020). Anti-CD123 CAR mRNA was synthesized in vitro and electroporated into expanded NK cells utilizing the Maxcyte electroporator. cam161533 TriKE was purified as previous described (Felices/Miller, Cancer Immunol Res. 2020). In vitro cytotoxicity against AML cells were evaluated at different E:T ratios (Chu/Cairo, JITC, 2020). Cytokines/growth factors were measured by ELISA assays and biolegend Legendplex assays. Results Anti-CD123 CAR expressed on more than 96% of expanded NK cells after CAR mRNA electroporation and lasted for about one week. Anti-CD123 CAR NK showed significantly enhanced in vitro cytotoxicity against CD123+ KG1a, HL-60 and MV4-11 with significantly enhanced IFN- 𝛾, granzyme B, and GM-CSF release compared to mock NK which did not express CAR (p&lt;0.001). The interaction between CAR NK and KG1a significantly enhanced Tim-3 and Galectin-9 expression. To prevent the AML escape from targeting, we examined the anti-tumor effect of the combination of anti-CD123 CAR NK with cam161533 TriKE. We found that cam161533 TriKE stimulated NK proliferation and significantly enhanced anti-CD123 CAR NK in vitro cytotoxicity against CD123+CD33+ MV4-11 cells compared to the one without TriKE (p&lt;0.05) at E:T=3:1. Conclusions The combination of anti-CD123 CAR NK and cam161533 TriKE had significant anti-AML effect. We will investigate the anti-AML effect of the combination utilizing human AML xenografts (Funded by DOD W81XWH-20-PRCRP-IPA #CA200119).
Citation Format: Alyssa S. Mendelowitz, Yaya Chu, Martin Felices, Jeffrey Miller, Stephen J. Forman, Gregory Behbehani, Challice Bonifant, Dean A. Lee, Mitchell Cairo. Anti-CD123 CAR NK cells and cam161533 Trispecific Killer Engager have synergistic activity against Acute Myeloid Leukemia [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P20.