330 Combination therapy with stem cell derived natural killer cells and monoclonal antibodies leads to potent ADCC through engagement of endogenouse CD16

Background

Glycostem’s ex vivo expansion and differentiation method in a fully closed automated manufacturing platform (uNiK™), generates GTA002 (oNKord®), an ‘off-the-shelf’ allogeneic cryopreserved NK cell product derived from umbilical cord blood CD34+ hematopoietic stem cell progenitor cells, which is currently being tested in a Phase I/II clinical trial in AML, WiNK (NCT04632316). Safety and tolerability of a non-cryopreserved predecessor was demonstrated in an earlier Phase I trial in AML (Dolstra et al. 2017). One of the important outcomes of this study was the notable increase in the CD16 expression of infused NK cells. Thus, we next exploited the potential of further enhancing and focusing cryopreserved NK cell anti-tumor responses in an antigen (Ag)-specific manner via antibody-dependent cellular cytotoxicity (ADCC) in pre-clinical models of hematological and solid malignancies.

Methods

Similar to its predecessor non-cryopreserved NK cells, GTA002 significantly upregulated CD16 expression in vivo in immunodeficient NCG mice. This spurred the optimization of the culture process to upregulate CD16 expression in order to study the ADCC potential of GTA002 in vitro. ADCC was assessed against CD19⁺ and HER2⁺ targets at low effector-to-target (E:T) ratios by end-point flow cytometry assays as well as impedance- and live imaging- (2D & 3D) based real time analysis. Next, we engineered CD16-NK cells by introduction of a lentiviral transduction step to the uNiK™ platform, to evaluate the effect of CD19-targeted ADCC of NK cells expressing engineered or endogenous CD16. Furthermore, expression of important activating and inhibitory receptors and intracellular levels of TNF, IFNγ, perforin and granzyme B were measured by flow cytometry to investigate their role in efficient cytotoxicity of GTA002 cells. We detected simultaneous tumor targeting by GTA002, both via preserved innate NK cell responses as well as Ag-specific targeting via ADCC at low E:T ratios.

Results

Moreover, GTA002 cells were tested for their ability to mediate killing of an ovarian cancer cell line in the presence of an Fc-active monoclonal antibody (mAb) targeting a tumor associated antigen expressed by SKOV-3 cells. Impedance-based cytotoxicity assays revealed that GTA002 exerted potent ADCC upon CD16 engagement. Addition of cytokine support further enhanced both baseline cytotoxicity as well as ADCC, leading to complete eradication of the SKOV-3 tumor cells.

Conclusions

Overall, the enhancement of the inherent potency of GTA002 by harnessing ADCC through combination therapy with mAbs achieved efficient Ag-specific responses demonstrating the great potential of multimodal targeting against a variety of challenging cancers using a highly safe ‘off-the-shelf’ NK cell-based cellular therapeutic.