Journal: Cell reports. Medicine

Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.

doi: 10.1016/j.xcrm.2025.102117

Figure Lengend Snippet: Figure 1. NKCEs redirect NK cells to kill target cells with minimal cytokine release (A) Design of tool molecules NCR1xHER2 (NKCE) and CD3xHER2 (TCE). (B) HER2 expression on JIMT1-Luc WT and HER2−/−. Antibodies bound per cell (ABCs) of HER2 are indicated in the graph. (C and D) JIMT1-Luc WT and HER2−/−were incubated with NK cells or Pan T cells at an E:T cell ratio of 5:1 and increasing concentrations of NCR1xHER2 or CD3xHER2. (C) JIMT1-Luc cell lysis at 24 h; concentrations of TNF and IFNγ (D) in the cell culture supernatants at 24 h. (E) Design of tool molecules NCR1xCLL1 (NKCE-I) and CD3xCLL1 (huCLL1 TCE). (F) CLL1 expression on PL21-Luc, NOMO1-Luc, and MOLM13-Luc. ABC of CLL1 is indicated in the graph. (G and H) PL21-Luc, NOMO1-Luc, and MOLM13-Luc were incubated with NK cells or Pan T cells at an E:T cell ratio of 2.5:1 and increasing concentrations of NKCE-I or huCLL1 TCE. (G) Target cell lysis at 48 h; (H) concentrations of TNF in the cell culture supernatants at 48 h. Data are presented as means ± SEM (C). The data shown are representative of at least two independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat#19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS

Techniques: Expressing, Incubation, Lysis, Cell Culture

Journal: Cell reports. Medicine

Article Title: Characterization and comparative analysis of multifunctional natural killer cell engagers during antitumor responses.

doi: 10.1016/j.xcrm.2025.102117

Figure Lengend Snippet: Figure 3. Multiple functional NKCEs induce robust killing activity with minimal TNF production (A) Components and designs of multifunctional NKCEs: NKCE-I to NKCE-V. (B–D) NOMO1-Luc cells were incubated with NK cells or Pan T cells at an E:T cell ratio of 2.5:1 and increasing concentrations of NKCE-I to NKCE-V and IL-2 or TCE. (B) NOMO1-Luc lysis at 48 h. EC50 and area under curve (AUC) are indicated in the graph. Concentrations of TNF (C) and IFNγ (D) in the cell culture su- pernatants at 48 h. (E) CLL1 expression on PL21-Luc WT and CLL1low. ABC of CLL1 is indicated in the graph. (F–H) PL21-Luc WT or CLL1low cells were incubated with NK cells at an E:T cell ratio of 2.5:1 and increasing concentrations of NKCE-III (F), NKCE-IV (G), and NKCE-V (H) and PL21-Luc lysis at 48 h. Data are presented as means ± SEM (B). The data shown are representative of at least two independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER muCD19 NKCE Amgen Inc In this paper muIgG-IL-2 Amgen Inc In this paper Critical commercial assays EasySepTM Human NK Cell Enrichment Kit STEMCELL Technologies Cat#19055 EasySepTM Human T cell Enrichment Kit STEMCELL Technologies Cat# 19051 EasySepTM Human CD4+ T cell Enrichment Kit STEMCELL Technologies Cat# 19052 EasySepTM Human CD8+ T cell Enrichment Kit STEMCELL Technologies Cat # 19053 Steady-Glo Luciferase Assay System Promega Cat# E2550 CellTiter-Glo® Luminescent Cell Viability Assay Promega Cat# G7573 CountBright Absolute Counting Beads Invitrogen Cat# C36950 BD Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences Cat# 554714 Foxp3 / Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 Live/Dead Fixable Near-IR Dead cell stain Kit Invitrogen Cat# L34975 CellTrace Violet Cell Proliferation Kit Invitrogen Cat# C34557 PE Phycoerythrin Fluorescence Quantitation Kit BD Biosciences Cat# 340495 U-PLEX CAR-T Cell Combo 1 (hu) Meso Scale Discovery (MSD) Cat# K15338K V-PLEX Human Proinflammatory Panel I Meso Scale Discovery (MSD) Cat# K15052D V-PLEX Proinflammatory Panel 1 Mouse Kit Meso Scale Discovery (MSD) Cat# K15048D V-PLEX Proinflammatory Panel 1 Human Kit Meso Scale Discovery (MSD) Cat# K15049D RNeasy Mini Kit Qiagen Cat# 74004 SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing Takara Bio Cat# 634890 Nextera XT DNA Library Preparation Kit Illumina Cat# FC-131-1096 Nextera XT Index Set D Illumina Cat# FC-131-2004 AMPure XP beads Beckman Coulter Cat# A63881 Deposited data Bulk RNA-Seq Amgen Inc GEO: GSE264122 Experimental models: Cell lines PL21 https://www.dsmz.de/ Cat# ACC 536 NOMO-1 https://www.dsmz.de/ Cat# ACC 542 MOLM13 https://www.dsmz.de/ Cat# ACC 554 JIMT1 https://www.dsmz.de/ Cat# ACC 589 MC38 ATCC Cat# CRL-2640 B16F10 ATCC Cat# CRL-6475 Experimental models: Organisms/strains B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) Taconic Cat# RAGN12-F C57BL/6N huCD3ε KI Amgen, breeding at CRL N/A NOD.Cg-Prkdcscid IL2Rgtm1Wjl/SzJ; NSG The Jackson Laboratory Strain# 005557 Oligonucleotides CLL1 sgRNA 1 IDT See Table S4 CLL1 sgRNA 2 IDT See Table S4 CLL1 sgRNA 3 IDT See Table S4 HER2 sgRNA 1 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 HER2 sgRNA 2 IDT See Table S4 NT sgRNA 1 IDT See Table S4 NT sgRNA 2 IDT See Table S4 (Continued on next page) Cell Reports Medicine 6, 102117, May 20, 2025 e3 Article ll OPEN ACCESS

Techniques: Functional Assay, Activity Assay, Incubation, Lysis, Cell Culture, Expressing