Preclinical Development of Inducible MyD88/CD40 (iMC)-Enhanced Chimeric Antigen Receptor Natural Killer (GoCAR-NK) Cells to Target BCMA+ Tumors
Authors: Wang Xiaomei , Duong MyLinh T , Guerrero Alan D. , Mahendravada Aruna , Sharp Kelly L , Brandt Mary E. , Gagliardi Christine , Foster Aaron E. , Bayle J. Henri 
Published: November 2020

Background: Natural Killer (NK) cells possess potent innate anti-tumor cytotoxicity that can be augmented and focused by engineering with chimeric antigen receptors (CARs). Because NK cells do not express T cell receptors that can direct alloreactivity, they have potential as an off-the-shelf (OTS) cell therapy for the treatment of cancer. We recently demonstrated that a drug-inducible co-activation molecule (inducible MyD88/CD40; iMC) synergizes with transgenic IL-15 to boost CAR-NK cell proliferation, survival and anti-tumor cytotoxic effects (Blood Adv.4:1950 [2020]). Here, we describe the pre-clinical development of an OTS iMC/IL-15-enhanced CAR-NK cell platform targeting B cell maturation antigen (BCMA) for the treatment of multiple myeloma.
Methods: NK cells were isolated from peripheral blood mononuclear cells by CD56+ selection, activated with IL-15 and microparticles conjugated with IL-21 and 4-1BB ligand. Activated NK cells were transduced with retrovirus encoding an optimized iMC and IL-15-expressing BCMA CAR construct (iMC-BCMA.z-IL15) where iMC signaling could be activated by exposure to rimiducid (Rim), a small molecule dimerizing ligand. Anti-tumor cytotoxicity and cytokine production was assessed using co-culture assays with control or modified CAR-NK cells against BCMA-expressing myeloma cells (NCIH929, RPMI8226, MM1S, U266 and NALM-6-BCMA). Additional experiments were performed with BCMA-edited cell lines (CRISPR/Cas9) to evaluate the innate cytotoxic potential of GoCAR-NK cells. In vivo anti-tumor efficacy and NK cell expansion was measured using immunodeficient NSG mice engrafted with 1.5 x 106 NCIH929-GFPffluc, MM1S-GFPffluc or THP1-GFPffluc cells followed by i.v. treatment with up to 1 x 107 BCMA GoCAR-NK cells. Tumor and NK cells were tracked via bioluminescence imaging.
Results: Following IL-15 and IL-21/4-1BBL microparticle stimulation, NK cells were efficiently transduced (40-70%) and exhibited rapid ex vivo expansion (200-fold in 13 days). iMC-BCMA.CAR-IL15-modified NK cells exhibited potent cytotoxicity against BCMA+target cells compared with mock-transduced NK cells (MM1S, 58±4% versus 17±2%; Nalm-6-BCMA, 61±2% versus 19±6%) after 24 hours. Long-term (7 day) co-culture assays revealed the effect of iMC/IL-15 enhancement on NK cell potency, proliferation and cytokine production where iMC-BCMA.z-IL15-modified NK cells stimulated with Rim showed a &gt;70% increase in tumor-specific killing compared to cells without iMC activation. Further, rimiducid-induced activation led to NK cell persistence and proliferation, 8.1±4.0-fold expansion compared to the start of the coculture. In comparison, there was an 80% reduction mock transduced NK cells or GoCAR-NK cells in cocultures without rimiducid. Induced-MC signaling also drove production of cytokines such as TNF-α, IFN-g (6.6X stimulation with 1 nM Rim relative to no drug), GMCSF, IP-10, and IL-13. In addition, activation of the iMC co-activation protein in combination with IL-15 secretion prevented NK cell exhaustion and led to retained functional activity of the modified GoCAR-NK cells for over 4-weeks in culture. In contrast, unmodified NK cells or modified GoCAR-NK cells without Rim exposure became functionally deficient. Of interest, a comparison of NK and T cells modified with the iMC/IL-15 BCMA CAR construct indicated that CAR-NK cells display more rapid target killing, which is further augmented by iMC-mediated cell signaling in the presence of Rim. Furthermore, GoCAR-NK cells were capable of lysis of BCMA-null target cells due to their innate anti-tumor activity. In vivo efficacy studies showed that neither iMC activation nor IL-15 secretion alone were sufficient to support CAR-NK cell engraftment in NSG mice but, in combination, they resulted in CAR-NK cell expansion and persistence. iMC/IL-15-enhanced BCMA GoCAR-NK cells proliferation was associated with improved control of tumor outgrowth in mice challenged with BCMA+ myeloma cells.
Summary: These results indicate that the synergistic activity of iMC signaling combined with transgenic IL-15 production can enhance BCMA-specific CAR-NK cytotoxicity, cytokine production, long-term proliferation and persistence and may improve overall anti-tumor efficacy of a potential OTS cell therapy for the treatment of myeloma.

Disclosures
Wang: Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Duong:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Guerrero:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Mahendravada:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharp:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Brandt:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gagliardi:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Foster:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Bayle:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.