Fig 2: 23ME-01473 significantly inhibits the growth of a NSCLC patient-derived xenograft cancer model in vivo.A, Cell surface expression of ULBP6/2/5 on EpCAM+ tumor cells from the CTG-3470 NSCLC PDX cell line implanted and grown in NOG mice as measured by flow cytometry. Data from three biological replicates are shown. B, Representative IHC images of ULBP6/2/5 or MICA/B expression (top) and the corresponding isotype controls (bottom) in CTG-3470 NSCLC PDX resected tumors. C, Soluble ULBP6/2/5 detected from the sera of three biological replicates. Data represent mean ± SD. D, Experimental design of in vivo efficacy study. E, Growth curves of NSCLC PDX tumors implanted in NOG mice treated with 10 mg/kg BIW 23ME-01473 or isotype control. Data represent mean ± SEM of 18 mice per group. Statistical analysis was performed using a one-sided test to determine if the ratio of mean adjusted area under the curve between 23ME-01473 and control groups was significantly less than 1, indicating lower tumor growth, with significance assessed at an FDR-adjusted alpha level of 0.05. Individual tumor spider plots for (F) isotype control or (G) 23ME-01473. Dotted lines indicate the day of NK cell infusion and the first and last doses of 23ME-01473 or isotype control. *, P ≤ 0.05. BIW, twice a week; EOS, end of study.

Fig 3: ULBP6 may be the most potent NKG2D ligand. A, Equilibrium dissociation constant KD for human NKG2DLs binding to NKG2D as measured by a Biacore surface plasmon resonance assay. Data represent mean ± SD of three technical replicates per condition. ULBP4 is not shown, as no binding to NKG2D was detected. B, IFNγ concentration of the supernatants of IL-2/IL-15–primed PBMCs cocultured with COV644 cells and 0.05 to 200 nmol/L recombinant sULBP6-02 or sMICA for 24 hours, depicted as a concentration-dependent response. No IFNγ was detected in COV644 cell supernatants without PBMC. Data represent mean ± SEM of three technical replicates per condition from one of three biological replicates. C, IFNγ concentration of the supernatants of IL-2/IL-15–primed PBMCs cocultured with 200 nmol/L plate-bound ULBP4 or MICA ± 250 nmol/L recombinant sULBP6-02 for 24 hours. Data represent mean ± SD of three technical replicates per condition from one of four biological replicates and were analyzed using one-way ANOVA for statistical significance. D, Cell surface NKG2D expression on human NK (CD45+ CD3– CD56+) cells or CD8+ T (CD45+ CD3+ CD56– CD8+) cells analyzed from IL-2/IL-15–primed PBMCs cultured with 50 nmol/L recombinant sULBP6-02 or PBS. Data represent mean ± SD of three technical replicates per condition from one of two biological replicates. Welch’s t test was used for statistical analysis. E, Quantification of COV644-GFP cell growth, as measured by GFP area per well by an Incucyte live cell analysis system, in the presence of IL-2/IL-15–primed PBMCs and 100 nmol/L recombinant sULBP6-02. Quantification is represented continuously over a 5-day time course. Data represent mean ± SD of three technical replicates per condition from one of four biological replicates. The last time point was analyzed using an unpaired t test for statistical significance. F, μMT−/− mice were inoculated with syngeneic MC38 EV or MC38 ULBP6-02 cells by subcutaneous injection (n = 10 mice per group). Nineteen days after inoculation, tumors were resected and processed for flow cytometric analysis. Bar graphs show the percentage of total tumor-infiltrating CD45+, NK (CD45+ Thy1.2– NK1.1+), NKT (CD45+ Thy1.2+ NK1.1+), and CD8+ T (CD45+ Thy1.2+ NK1.1– CD8+) cells and the percentage of CD25+ NK and NKT cells. Data represent mean ± SEM from one of two independent experiments and were analyzed using Welch’s t test for statistical significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. ns, not significant.

Fig 4: Cytokine secretion, cytolytic activity and proliferation of CD19/NKG2DL tandem CAR T-cells co-cultured with CD19+ and CD19- Nalm-6 cells. (A) Expression of CD19 and NKG2DL in Nalm-6 and Nalm-6 CD19 KO cells. (B) Expression of individual NKG2DL using antibodies directed against MICA, MICB, ULBP1, ULBP3 or ULBP2-5-6 (grey histograms) vs autofluorescence (white histograms). (C) Secretion of IFN-γ, TNF-α and IL-2 cytokines after a 24-hour co-culture at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells. (D) Cytolytic activity of CAR T-cells against Nalm-6 and Nalm-6 CD19 KO cells at 1:1 and 0.1:1 E:T ratio. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (E) Representative experiment showing CTV histograms of CAR T-cells after 4 days of co-culture without cancer cells or at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells (4 individual experiments were performed on 4 different donors). Adjusted P values (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data presented as means (SD) of n=5. Each symbol denotes a different PBMC donor.

Fig 5: Cytokine secretion of CD19/NKG2DL tandem CAR T-cells upon stimulation with coated CD19 or coated MICA. IFN-γ secretion after a 24-hour exposure with increasing concentrations of (A) coated rhCD19-Avi or (B) coated rhMICA-Fc.