Engineering High Affinity and Cleavage Resistant CD16 to Augment ADCC of Placental Hematopoietic Stem Cells-Derived Natural Killer Cells

Background:

Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the-shelf, ex-vivo expanded and allogenic natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified. The 158Val/Val form has shown to have higher IgG binding affinity compared to the 158Phe/Phe form.¹ The high IgG binding allele are found in about 10-20% of the normal population.^2,3 In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells.⁴ Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity.

Methods:

Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3x10⁶) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10x10⁶) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism.

Results:

Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells expanded [7095 ± 2998 folds (n=8)] and differentiated into PNK cells (>90% CD56+CD3-) at day 35. PNK-CD16VP expressed 64.6 ± 10.3% (n=8) of CD16, while the NT expressed 12.1 ± 3.3% (n=8) CD16. PMA/ionomycin induced >89% shedding of CD16 in NT cells, while significantly less (<11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 47 ± 13% against Daratumumab opsonized Daudi cells versus 25 ± 5% (n=5; p<0.05); 30 ± 13% against Daratumumab opsonized HS-Sultan cells versus 21 ± 14% (n=3; p<0.05); 30 ± 7% against Daratumumab opsonized Sudhl6 cells versus 16 ± 10% (n=3; p<0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji and Sudhl4 with Daratumumab and Rituximab antibodies.

PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal (>50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity.

Conclusions:

In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted.

References:

1. Wu J et al. J Clin Invest. 1997;100(5):1059-1070.

2. Sugita N et al. Clin Exp Immunol. 1999;117(2):350-354.

3. Koene HR et al. Blood. 1997;90(3):1109-1114.

4. Jing Y et al. PLoS One. 2015;10(3):e0121788.