Background B7H3 is a tumor associated antigen (TAA), found on numerous malignancies including prostate, lung, and breast cancers. High levels of B7H3 expression are correlated with late stage disease and poor prognosis. Furthermore, B7H3 is minimally expressed on normal tissue, making it an ideal TAA for broad cancer treatment strategy. We developed a tri-specific killer engager (TriKETM) consisting of a nanobody anti-CD16, IL-15, and nanobody anti-B7H3 joined by flexible linkers (camB7H3 TriKE) (figure 1A). The combination of B7H3 TriKE with an off-the-shelf NK cell therapy presents an appealing therapeutic strategy for the treatment of solid tumors with decreased risk of toxicity in allogeneic settings compared to T-cell derived products.

Methods An anti-B7H3 nanobody was developed via biopanning and cloned into a TriKE vector. TriKE was produced in Expi293 cells and affinity purified using poly-His tag. NK cells were co-incubated with cell lines exhibiting a range of B7H3 expression and with 3nM of camelid B7H3 TriKE or control. We have previously derived NK cells expressing high affinity non-cleavable hnCD16, CD38 KO, and IL-15/IL-15R fusion from clonal master engineered iPSC lines. Engineered iNK cells were tested in conjunction with the TriKE. A repeated measures ANOVA was used for statistical comparisons as noted in figure legends

Results Engineered iNK cells co-incubated with camB7H3 TriKE and C4-2 prostate cells significantly increased degranulation (CD107a) and cytokine production (IFN-gamma) compared to controls (figure 1B/C, P<0.05, n=3). camB7H3 TriKE directly bound C4-2 cells with an estimated EC50 of approximately 3nM. camB7H3 TriKE increased percentages of engineered iNK cells dividing robustly (3 or more times) compared to corresponding IL-15 doses at 3 nM (figure 1D, P<0.001, n=3). Furthermore, camB7H3 TriKE enhanced cytotoxic activity of engineered iNK cells against a variety of tumor cells in 2D and spheroid format independent of cytokine support (figure 1E-F). Engineered iNK cells incorporating an anti-B7H3 chimeric antigen receptor (CAR) is also being developed and will be discussed.

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Abstract 470 Figure 1

A) Schematic of TriKE molecule demonstrating spatial relationship of anti-CD16 nanobody, IL-15, and anti-B7H3 nanobody with flexible linker regions. B) Percent of PB NK cells CD107a as a marker of NK degranulation. Unselected PBMCs were stimulated with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment with B7H3-expressing C4-2 prostate cancer cell lines Cells were stimulated for 5 hours and evaluated for degranulation (surface CD107a) by flow cytometry, gating on the NK (CD56+CD3-) population (displayed). Graphs display mean ± SEM. P<0.05 using repeated measures ANOVA. C) Percent of PB NK cells expressing intracellular interferon-gamma. Unselected PBMCs were stimulated with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment with B7H3-expressing C4-2 prostate cancer cell lines Cell were stimulated for 5 hours and evaluated for degranulation (surface CD107a) by flow cytometry, gating on the NK (CD56+CD3-) population (displayed). Graphs display mean ± SEM. P<0.05 using repeated measures ANOVA. D) Percent of PB NK cells dividing robustly (3 or more times) over a 7 day stimulation with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment. PBMCs were incubated with Cell-Trace Violet reagent prior to stimulation. Graphs display mean ± SEM. P<0.001 using repeated measures ANOVA. E) Representative 2D IncuCyte images of PC3 prostate cancer cell lines transduced with NucLight Red. Cells were co-incubated with iNK alone, iNK with 3nM IL-15 or iNK with 3 nM antiB7H3 TriKE. Images represent remaining NucLight Red transduced PC3 cells after 72 hours of co-incubation as noted above. F) Representative microspheriod IncuCyte images of PC3 prostate cancer lines transduced with NucLight Red. Cells were co-incubated with iNK cells alone, iNK with 3 nM IL-15 or iNK with 3 nM antiB7H3 TriKE. Images represent remaining NucLight Red transduced PC3 cells after 72 hours of co-incubation as noted above

Conclusions camB7H3 TriKE dramatically increases function and activation on endogenous NK cells as well as engineered iNK cell, which can be adoptively transferred to patients with a broad range of cancers, including prostate cancer. TriKE activity was potent across a broad concentration spectrum and corresponded directly with B7H3 target expression. These studies represent the proof-of-concept of a novel pairing of off-the-shelf, engineered iNK cells with B7H3-directed pan-cancer engager molecules (TriKEs and CARs) to enhance specificity, persistence and anti-tumor function.