nkg2d cd314  (Novus Biologicals)

Journal: bioRxiv

Article Title: Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

doi: 10.64898/2026.02.09.704789

Figure Lengend Snippet: Molecular characterization and purification of NaMiX and Engagers. (A) The molecular pattern of NaMiX, TriKE and control BiKEs was analyzed by Western Blot under non-reducing (NR) or reducing (R) conditions and revealed with anti-His antibody coupled to AlexaFluor 488. (B) NaMiX and TriKE were purified using His affinity chromatography and TriKE was further purified using StrepXT affinity chromatography. Binding of NaMiX (D), TriKE (E), BiKE NKG2D (F) and BiKE NKp30 (G) was evaluated on HEK-293T (CEA - NKG2D - NKp30 - ), BxPC-3 (CEA + ), NK92-CD16 (NKp46 + ) and on KHYG-1 (NKG2D + NKp30 + ) cell lines and detected by anti-His antibody by flow cytometry. (E-F) Binding of NaMiX, TriKE and BiKEs was also assessed by ELISA. (H) For NaMiX, recombinant human NKp46 (rNKp46) was coated then NaMiX was added in presence (red) or in absence (green) of blocking antibody and revelation was performed using anti-6x his antibody conjugated to HRP. For TriKE and BiKEs, ELISA assays were performing using rNKG2D, rNKp30 or rCEA as coating, then adding the engager of interest in presence or absence of adequate blocking antibody (anti-NKG2D, anti-NKp30 or anti-CEA) then revealing with anti-6x his antibody conjugated to HRP. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (2-3 replicates per experiment).

Article Snippet: For binding assays, 1 μg/mL of the appropriate recombinant (r) protein rNKp46 (R&D Systems, #1850-NK-025), rNKp30 (Sinobiological, #10480-H02H), rNKG2D (Sinobiological, #10575-H01S) or rCEACAM5 (rCEA) (R&D systems, #10449-CM) was coated on a MaxiSorp 96-well flat-bottom ELISA plate (ThermoScientific, # 442404) overnight.

Techniques: Purification, Control, Western Blot, Affinity Chromatography, Binding Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay

Journal: bioRxiv

Article Title: Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

doi: 10.64898/2026.02.09.704789

Figure Lengend Snippet: (A-B) ELISA assay using recombinant human CEA (rCEA) as coating, adding TriKE, then revealing by (A) recombinant NKG2D coupled to Fc (rNKG2D-Fc) or (B) recombinant NKp30 coupled to Fc (rNKp30-Fc) and an anti-Fc linked with HRP. (C-D) CFSE-stained BxPC-3 (green) and CellTracker Deep Red-stained KHYG-1 (red/pink) cells were co-cultured with (C) control medium or with TriKE and analyzed by fluorescence microscopy or (D) with AlexaFluor647-labeled TriKE (red) and analyzed by confocal microscopy. (E-G) CFSE-stained BxPC-3 were seeded in ultra-low attachment plates for 48 hours to form spheroids, then co-incubated with CellTracker Deep Red stained PBMCs pre-incubated with NaMiX or control medium for 48 hours, together with BiKEs, TriKE, control scaffold or control medium and placed inside IncucyteS3 for 96 hours. Spheroid size was assessed using ImageJ software and the percentage of initial spheroid size is represented (E) after 48 hours of co-incubation or (F) over 96 hours of co-incubation. (G) Representative image at day 0, day 3 and day 4 of co-incubation. (H-I) Representative images of CFSE-stained BxPC-3 co-incubated with purified NK cells from a healthy donor (H) and a PDAC patient (I) and the cytotoxicity marker DRAQ7 at day 3 of co-incubation with indicated molecules. Data are presented as the mean values ± SEM. Results correspond to 2 pooled representative donors. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p<0.05; **p<0.01; ***p<0,001). Scale bar = 400 µm.

Article Snippet: For binding assays, 1 μg/mL of the appropriate recombinant (r) protein rNKp46 (R&D Systems, #1850-NK-025), rNKp30 (Sinobiological, #10480-H02H), rNKG2D (Sinobiological, #10575-H01S) or rCEACAM5 (rCEA) (R&D systems, #10449-CM) was coated on a MaxiSorp 96-well flat-bottom ELISA plate (ThermoScientific, # 442404) overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Staining, Cell Culture, Control, Fluorescence, Microscopy, Labeling, Confocal Microscopy, Incubation, Software, Purification, Marker

Journal: bioRxiv

Article Title: Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

doi: 10.64898/2026.02.09.704789

Figure Lengend Snippet: (A-B) ELISA assay using recombinant human CEA (rCEA) as coating, adding BiKE, then revealing by (A) recombinant NKG2D coupled to Fc (rNKG2D-Fc) or (B) recombinant NKp30 coupled to Fc (rNKp30-Fc) and an anti-Fc linked with HRP. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (3 replicates per experiment). (B) PBMCs were incubated with BiKE, TriKE or control scaffold C4BPβ together with BxPC-3 cells at an E:T ratio of 10:1 for 24 hours and the cell culture supernatant was analyzed for IFN-γ by ELISA. Data is represented by individual donor (n=3-5). (C-D) CFSE-stained BxPC-3 cells were seeded in ultra-low attachment plates for 48 hours, then co-incubated with CellTracker Deep Red stained PBMCs (blue) and BiKEs, TriKE, control scaffold C4BPβ or control medium and placed inside IncucyteS3 for 96 hours. Pictures were acquired every 6 hours and spheroid size was quantified using ImageJ software. Scale bar = 400 µm.

Article Snippet: For binding assays, 1 μg/mL of the appropriate recombinant (r) protein rNKp46 (R&D Systems, #1850-NK-025), rNKp30 (Sinobiological, #10480-H02H), rNKG2D (Sinobiological, #10575-H01S) or rCEACAM5 (rCEA) (R&D systems, #10449-CM) was coated on a MaxiSorp 96-well flat-bottom ELISA plate (ThermoScientific, # 442404) overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Control, Cell Culture, Staining, Software

Journal: bioRxiv

Article Title: Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

doi: 10.64898/2026.02.09.704789

Figure Lengend Snippet: (A-B) Both NaMiX and TriKE expressed dimers upstream and downstream of the dimerization domain of the C4 binding protein β (C4BP-β). NaMiX is composed of IL-15Rα/IL-15 complexes associated to anti-NKp46 single chain variable fragments (scFvs) and TriKE and BiKEs are composed of scFvs targeted against NKG2D and/or NKp30 and VHH against CEA. PBMCs were pre-incubated with NaMiX, rhu-IL15 or control medium for 48 hours, and BxPC-3 cells were added for 5 additional hours at an effector:target ratio of 10:1. (C) NK cells were then analyzed by flow cytometry for their expression of CD107a, IFN-γ, perforin and granzyme B. (D-E) Representative dot plots for CD107a and IFN-γ expression and histograms for perforin and granzyme B expression. (F) The cell culture supernatant was analyzed for perforin, granzyme B and IFN-γ by ELISA. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (2 donors per experiment). Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p<0.05; **p<0.01).

Article Snippet: Then, 5 μg/mL TriKE or BiKEs was added to the plate, which was revealed with either Fc-tagged recombinant NKG2D (rNKG2D) (Sinobiological, #10575-H01S) or recombinant NKp30 (rNKp30) (Sinobiological, #10480-H02H) followed by goat anti-human Fc-HRP (Invitrogen, #A18817).

Techniques: Binding Assay, Incubation, Control, Flow Cytometry, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

doi: 10.64898/2026.02.09.704789

Figure Lengend Snippet: Molecular characterization and purification of NaMiX and Engagers. (A) The molecular pattern of NaMiX, TriKE and control BiKEs was analyzed by Western Blot under non-reducing (NR) or reducing (R) conditions and revealed with anti-His antibody coupled to AlexaFluor 488. (B) NaMiX and TriKE were purified using His affinity chromatography and TriKE was further purified using StrepXT affinity chromatography. Binding of NaMiX (D), TriKE (E), BiKE NKG2D (F) and BiKE NKp30 (G) was evaluated on HEK-293T (CEA - NKG2D - NKp30 - ), BxPC-3 (CEA + ), NK92-CD16 (NKp46 + ) and on KHYG-1 (NKG2D + NKp30 + ) cell lines and detected by anti-His antibody by flow cytometry. (E-F) Binding of NaMiX, TriKE and BiKEs was also assessed by ELISA. (H) For NaMiX, recombinant human NKp46 (rNKp46) was coated then NaMiX was added in presence (red) or in absence (green) of blocking antibody and revelation was performed using anti-6x his antibody conjugated to HRP. For TriKE and BiKEs, ELISA assays were performing using rNKG2D, rNKp30 or rCEA as coating, then adding the engager of interest in presence or absence of adequate blocking antibody (anti-NKG2D, anti-NKp30 or anti-CEA) then revealing with anti-6x his antibody conjugated to HRP. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (2-3 replicates per experiment).

Article Snippet: Then, 5 μg/mL TriKE or BiKEs was added to the plate, which was revealed with either Fc-tagged recombinant NKG2D (rNKG2D) (Sinobiological, #10575-H01S) or recombinant NKp30 (rNKp30) (Sinobiological, #10480-H02H) followed by goat anti-human Fc-HRP (Invitrogen, #A18817).

Techniques: Purification, Control, Western Blot, Affinity Chromatography, Binding Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay

Journal: bioRxiv

Article Title: Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

doi: 10.64898/2026.02.09.704789

Figure Lengend Snippet: NaMiX stimulates the degranulation and activation of NK cells via STAT5 but not mTOR pathway. Healthy donors PBMCs were pre-incubated with NaMiX, rhu-IL15 or control medium for 48 hours. PBMCs were stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (to exclude dead cells), anti-human CD3 (to exclude CD3 + T cells), anti-human CD14 and CD19 (to exclude B cells and monocytes), and with anti-human CD56 and CD16 to identify NK cells. (A) Gating strategy. (B) NK cells were then analyzed by flow cytometry for their expression of CD107a, IFN-γ and perforin. (C) The cell culture supernatant was analyzed for perforin, granzyme B and IFN-γ by ELISA. (D) PBMCs were incubated with NaMiX for 5 minutes then NK cells were analyzed by flow cytometry for their expression of p-STAT5 and p-rpS6. (E-F) PBMCs pre-incubated with NaMiX for 48 hours were studied for their expression of Ki67, NKG2D and NKp30 on NK cells by flow cytometry. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (1-3 donors per experiment). Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Article Snippet: Then, 5 μg/mL TriKE or BiKEs was added to the plate, which was revealed with either Fc-tagged recombinant NKG2D (rNKG2D) (Sinobiological, #10575-H01S) or recombinant NKp30 (rNKp30) (Sinobiological, #10480-H02H) followed by goat anti-human Fc-HRP (Invitrogen, #A18817).

Techniques: Activation Assay, Incubation, Control, Staining, Flow Cytometry, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

doi: 10.64898/2026.02.09.704789

Figure Lengend Snippet: (A-B) ELISA assay using recombinant human CEA (rCEA) as coating, adding TriKE, then revealing by (A) recombinant NKG2D coupled to Fc (rNKG2D-Fc) or (B) recombinant NKp30 coupled to Fc (rNKp30-Fc) and an anti-Fc linked with HRP. (C-D) CFSE-stained BxPC-3 (green) and CellTracker Deep Red-stained KHYG-1 (red/pink) cells were co-cultured with (C) control medium or with TriKE and analyzed by fluorescence microscopy or (D) with AlexaFluor647-labeled TriKE (red) and analyzed by confocal microscopy. (E-G) CFSE-stained BxPC-3 were seeded in ultra-low attachment plates for 48 hours to form spheroids, then co-incubated with CellTracker Deep Red stained PBMCs pre-incubated with NaMiX or control medium for 48 hours, together with BiKEs, TriKE, control scaffold or control medium and placed inside IncucyteS3 for 96 hours. Spheroid size was assessed using ImageJ software and the percentage of initial spheroid size is represented (E) after 48 hours of co-incubation or (F) over 96 hours of co-incubation. (G) Representative image at day 0, day 3 and day 4 of co-incubation. (H-I) Representative images of CFSE-stained BxPC-3 co-incubated with purified NK cells from a healthy donor (H) and a PDAC patient (I) and the cytotoxicity marker DRAQ7 at day 3 of co-incubation with indicated molecules. Data are presented as the mean values ± SEM. Results correspond to 2 pooled representative donors. Statistical analysis was performed using a one-way ANOVA and post-hoc Tukey test (*p<0.05; **p<0.01; ***p<0,001). Scale bar = 400 µm.

Article Snippet: Then, 5 μg/mL TriKE or BiKEs was added to the plate, which was revealed with either Fc-tagged recombinant NKG2D (rNKG2D) (Sinobiological, #10575-H01S) or recombinant NKp30 (rNKp30) (Sinobiological, #10480-H02H) followed by goat anti-human Fc-HRP (Invitrogen, #A18817).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Staining, Cell Culture, Control, Fluorescence, Microscopy, Labeling, Confocal Microscopy, Incubation, Software, Purification, Marker

Journal: bioRxiv

Article Title: Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

doi: 10.64898/2026.02.09.704789

Figure Lengend Snippet: (A-B) ELISA assay using recombinant human CEA (rCEA) as coating, adding BiKE, then revealing by (A) recombinant NKG2D coupled to Fc (rNKG2D-Fc) or (B) recombinant NKp30 coupled to Fc (rNKp30-Fc) and an anti-Fc linked with HRP. Data are presented as the mean values ± SEM. Results correspond to two pooled independent experiments (3 replicates per experiment). (B) PBMCs were incubated with BiKE, TriKE or control scaffold C4BPβ together with BxPC-3 cells at an E:T ratio of 10:1 for 24 hours and the cell culture supernatant was analyzed for IFN-γ by ELISA. Data is represented by individual donor (n=3-5). (C-D) CFSE-stained BxPC-3 cells were seeded in ultra-low attachment plates for 48 hours, then co-incubated with CellTracker Deep Red stained PBMCs (blue) and BiKEs, TriKE, control scaffold C4BPβ or control medium and placed inside IncucyteS3 for 96 hours. Pictures were acquired every 6 hours and spheroid size was quantified using ImageJ software. Scale bar = 400 µm.

Article Snippet: Then, 5 μg/mL TriKE or BiKEs was added to the plate, which was revealed with either Fc-tagged recombinant NKG2D (rNKG2D) (Sinobiological, #10575-H01S) or recombinant NKp30 (rNKp30) (Sinobiological, #10480-H02H) followed by goat anti-human Fc-HRP (Invitrogen, #A18817).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Control, Cell Culture, Staining, Software