Journal: Neuro-Oncology Advances

Article Title: Dual targeting of CD155 / TIGIT and PD-L1 / PD-1 immune checkpoints potentiates NK cell-mediated cytotoxicity in medulloblastoma

doi: 10.1093/noajnl/vdaf099

Figure Lengend Snippet: Primary MB show abundant CD155 expression and TIGIT + immune cell infiltrate. (a) Online R2 genomics analysis and visualization platform was used for gene expression of CD155 ( PVR, left) and TIGIT (right). On the left of each graph the entire MB cohort (Cavalli, n = 763) is shown and on the right expression per MB subgroup is shown (WNT: n = 70, SHH: n = 223, G3: n = 144, G4: n = 326). Independent cohort of MB patients was analyzed for CD155 mRNA (b, left) and protein (g) and TIGIT mRNA (b, right) and protein (h) (mRNA cohort n = 48, protein cohort n = 45). Single-cell transcriptomics clustering for molecular subtype (c, left) and cell type (c, right) was performed with subsequent analysis of the lymphoid compartment (d). Immune checkpoints protein mRNA expression was checked in (e) and investigated per cell type in (f). One-way ANOVA with Holm-Šidák’s multiple comparisons test (a) was used for statistical analysis; ** P < .01, *** P < .001, **** P < .0001. Statistical analysis was performed automatically in the R2 platform. (g,h) Scale bars represent 250 µm. Abbreviations: G3, Group 3; G4, Group 4; MB, Medulloblastoma; Neg Ctrl, Negative control (medulloblastoma tissue); Pos Ctrl, Positive control (tonsil tissue); SHH, Sonic hedgehog; WNT, Wingless.

Article Snippet: Tissue slides were deparaffinized, blocked, cooked in EDTA, and stained by primary antibodies against PD-L1 (Roche, Ready-to-use (RTU) clone SP263, J17387), CD3 (Roche, RTU, 2GV6, K10044), and CD155 (Cell Signaling clone D3G7H, 13544S, 1:500) was performed using an automated immunostainer (Benchmark Ultra, Ventana, Roche) using the OptiView DAB detection kit (REF: 760-700) and UltraVIEW Universal Alkaline Phosphatase Red Detection Kit (REF: 760-501).

Techniques: Expressing, Gene Expression, Single-cell Transcriptomics, Negative Control, Positive Control

Journal: Neuro-Oncology Advances

Article Title: Dual targeting of CD155 / TIGIT and PD-L1 / PD-1 immune checkpoints potentiates NK cell-mediated cytotoxicity in medulloblastoma

doi: 10.1093/noajnl/vdaf099

Figure Lengend Snippet: CD155 / TIGIT blockade potentiates NK cell-mediated killing of MB cells. Baseline mRNA and protein expression levels of CD155 were examined for MB cell lines by qPCR (a), western blot (b), flow cytometry (c), and confocal microscopy (d); all n = 3. For NK cells, baseline mRNA and protein expression levels of TIGIT were examined by qPCR (e), western blot (f), and flow cytometry (g); all n = 3. In flow cytometry plots, green = unstained, yellow = isotype, and purple = anti- CD155 (c) or anti- TIGIT (g). (h) TCR-transduced Jurkat cells were cultured and TCR was activated by adding CD3/CD28 activation beads (+/- ‘Beads’). MB cells were added with or without anti- TIGIT to examine reduction and recovery in TCR activation, respectively; n = 4. (i) Baseline killing of Daoy (control, left) and MB cell lines D283 (middle) and HD-MB03 (right) by NK-92 in different E:T ratios; all n ≥ 3. (j,k) Effect of Tiragolumab treatment on the killing of Daoy (left) and MB cell lines D283 (middle), and HD-MB03 (right) by NK-92 at E:T 1:1 (j) and 2:1 (k); n ≥ 3. For (a,e,h–k), data is shown as mean ± 95% confidence interval (CI), and different colors correspond to multiple biological replicates. One-way ANOVA with Holm-Šidák’s multiple comparisons test (h–i) and two-tailed paired t -test (j,k) were used for statistical analysis, * P < .05, ** P < .01, *** P < .001, **** P < .0001. Abbreviation: dCT, delta CT.

Article Snippet: Tissue slides were deparaffinized, blocked, cooked in EDTA, and stained by primary antibodies against PD-L1 (Roche, Ready-to-use (RTU) clone SP263, J17387), CD3 (Roche, RTU, 2GV6, K10044), and CD155 (Cell Signaling clone D3G7H, 13544S, 1:500) was performed using an automated immunostainer (Benchmark Ultra, Ventana, Roche) using the OptiView DAB detection kit (REF: 760-700) and UltraVIEW Universal Alkaline Phosphatase Red Detection Kit (REF: 760-501).

Techniques: Expressing, Western Blot, Flow Cytometry, Confocal Microscopy, Cell Culture, Activation Assay, Control, Two Tailed Test

Journal: Neuro-Oncology Advances

Article Title: Dual targeting of CD155 / TIGIT and PD-L1 / PD-1 immune checkpoints potentiates NK cell-mediated cytotoxicity in medulloblastoma

doi: 10.1093/noajnl/vdaf099

Figure Lengend Snippet: Dual targeting of CD155 / TIGIT and PD-L1 / PD-1 enhance primary NK cell activation and killing towards MB cells and organoids. Therapy of Tiragolumab (left) and Durvalumab (right) compared to isotype increases IFN-γ + primary NK cells in Daoy (a), D283 (b), and HD-MB03 (c) co-cultures, all n ≥ 3. Combination therapy of Tiragolumab and Durvalumab on Daoy (d) in E:T = 2:1 and IFN-γ primed D283 (e, left), and HD-MB03 (e, right) in E:T = 1:1, all n ≥ 3. (f) Microscopic image of 3D organoid structure in culture. Scale bars represent 500 μm. (g) Baseline expression levels of CD155 (left) and PD-L1 (right), both n = 3. In flow cytometry plots, yellow = isotype and purple = anti- CD155 or anti- PD-L1 . (h) IFN-γ- and TNF-α-induced upregulation of PD-L1 expression ( n = 3) and (i) baseline killing of MED113FH (left) and B062-008 (right) by primary NK cells, n = 4. Combination therapy of Tiragolumab and Durvalumab in an E:T ratio of 1:1 in MED113FH (j, left) and B062-008 (j, right and k) without (j) and with (k) priming by IFN-γ to induce PD-L1 expression, all n = 4. (l, left) Representative examples of real-time imaging screenshots from co-cultures of primary NK cells and B062-008 organoids taken every four hours. Green = alive B062-008 organoids, red = dead B062-008 organoids, blue = primary NK cells, white bars represent 100 µm, n = 4. (l, middle) Baseline killing of B062-008 by primary NK cells measured by real-time imaging using cumulative cell death analysis, n = 4. (l, right) Donor-specific effects on single and combination treatment of Tiragolumab and Durvalumab measured by real-time imaging using cumulative cell death analysis, n = 4. Data (d,e,h–k) is shown as mean ± 95% confidence interval (CI). For (a–c,l), different colors correspond to different NK cell donors, and for (d,e,h–k) different colors correspond to multiple biological replicates. One-way ANOVA with Holm-Šidák’s multiple comparisons test (d,e,h,j,k) and two-tailed paired t -test (a–c,i) were used for statistical analysis, * P < .05, ** P < .01, *** P < .001, **** P < .0001. Abbreviation: IFN-γ, Interferon-γ; TNF-α, Tumor necrosis factor-α.

Article Snippet: Tissue slides were deparaffinized, blocked, cooked in EDTA, and stained by primary antibodies against PD-L1 (Roche, Ready-to-use (RTU) clone SP263, J17387), CD3 (Roche, RTU, 2GV6, K10044), and CD155 (Cell Signaling clone D3G7H, 13544S, 1:500) was performed using an automated immunostainer (Benchmark Ultra, Ventana, Roche) using the OptiView DAB detection kit (REF: 760-700) and UltraVIEW Universal Alkaline Phosphatase Red Detection Kit (REF: 760-501).

Techniques: Activation Assay, Expressing, Flow Cytometry, Imaging, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: COM902, a novel therapeutic antibody targeting TIGIT augments anti-tumor T cell function in combination with PVRIG or PD-1 pathway blockade

doi: 10.1007/s00262-021-02921-8

Figure Lengend Snippet: COM902 does not induce ADCC or CDC activity. a ADCC activity assessment for COM902. b CDC activity assessment for COM902. Campath (anti-CD52) hIgG1 was used as a positive control. Representative data from n ≥ 2 is shown for each graph

Article Snippet: For blocking experiments, the cell lines described above were pre-incubated with COM902 or hIgG4 isotype for 30 min. Recombinant hPVR-mIgG2a-Fc (hPVR-Fc) and cynomolgus PVR-hIgG1-Fc-biotin (cPVR-Fc) produced internally, or mouse PVR-hIgG1-Fc (mPVR-Fc) (Sino Biological) were added to the cells for 1 h and detected with fluorescently labeled secondary antibodies.

Techniques: Activity Assay, Positive Control