Journal: PLOS One

Article Title: Inhibition of HER2 signaling and breast cancer cell growth with a novel antibody targeting HER2 ECD III/IV

doi: 10.1371/journal.pone.0338127

Figure Lengend Snippet: A. Competitive ELISA assessing the binding of His-tagged IgGs to immobilized HER2 in the presence or absence of saturating concentrations of non-His-tagged IgGs, detected with an anti-His-HRP antibody. B. Saturation binding curves of trastuzumab and pertuzumab to HER2 on live MCF7 cells. Cells were incubated with antibodies (0-400 nM) for 30 min and detected with an AF647-conjugated goat anti-human secondary antibody via flow cytometry. C. Competitive binding of of AF647-labeled trastuzumab or pertuzumab (0-300 nM) to MCF7 cells pre-saturated with 267 nM of unlabeled trastuzumab and pertuzumab, analyzed by flow cytometry. D. Binding of AF647-conjugated m66 or m75 to MCF7 cells after pre-incubation with 267 nM trastuzumab or pertuzumab. E. Binding of AF647-conjugated rabbit antibodies to MCF7 cells pre-saturated with 267 nM of trastuzumab and pertuzumab, evaluated by flow cytometry. T, trastuzumab; P, pertuzumab. Error bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, T-test.

Article Snippet: For ligand-dependent HER2 signaling, cells were first incubated with 10 μg/mL each antibody or combinations for 24 h, followed by stimulation with 0.5 nM recombinant human NRG1 or 5 nM EGF (Acro Biosystems, EGF-H52H3) for 10 min. After washing with ice-cold PBS, the cells were lysed, and the cell lysates were separated by 4–20% gradient SDS-PAGE under reducing conditions.

Techniques: Competitive ELISA, Binding Assay, Incubation, Flow Cytometry, Labeling

Journal: PLOS One

Article Title: Inhibition of HER2 signaling and breast cancer cell growth with a novel antibody targeting HER2 ECD III/IV

doi: 10.1371/journal.pone.0338127

Figure Lengend Snippet: A. Viability of BT-474 cells following 4 d treatment with a 1:3 serial dilution (0-100 nM) of the indicated antibodies, as assessed by CCK-8 assay. B. Dose-dependent inhibition of SK-BR-3 and MCF7 cell proliferation after 4 d of exposure to antibody dilutions (0-100 nM). Data are presented as mean ± SEM from n = 5 (BT-474 and SK-BR-3) and n = 6 (MCF7) independent experiments. C. SPRi analysis of the binding kinetics between HER2 and trastuzumab, pertuzumab, m66 and r40. Antibodies (0.5 mg/mL) were immobilized on a 3D optical crosslinking biosensor chip. HER2 was injected at concentrations ranging from 1 to 128 nM. Association and dissociation phases were monitored for 285 s and 930 s, respectively, using a PlexArray ® HT system.

Article Snippet: For ligand-dependent HER2 signaling, cells were first incubated with 10 μg/mL each antibody or combinations for 24 h, followed by stimulation with 0.5 nM recombinant human NRG1 or 5 nM EGF (Acro Biosystems, EGF-H52H3) for 10 min. After washing with ice-cold PBS, the cells were lysed, and the cell lysates were separated by 4–20% gradient SDS-PAGE under reducing conditions.

Techniques: Serial Dilution, CCK-8 Assay, Inhibition, Binding Assay, Injection

Journal: PLOS One

Article Title: Inhibition of HER2 signaling and breast cancer cell growth with a novel antibody targeting HER2 ECD III/IV

doi: 10.1371/journal.pone.0338127

Figure Lengend Snippet: A-C. Immunoblots showing HER2 downstream signaling in (A) MCF7, (B) BT-474 and (C) SK-BR-3 cells treated for 24 h with 10 μg/mL of the indicated antibodies. Whole-cell lysates were immunoblotted with antibodies against the indicated proteins. For combination treatments, the indicated concentration refers to each antibody individually. The numerical values shown in the figures represent normalized quantitative data.

Article Snippet: For ligand-dependent HER2 signaling, cells were first incubated with 10 μg/mL each antibody or combinations for 24 h, followed by stimulation with 0.5 nM recombinant human NRG1 or 5 nM EGF (Acro Biosystems, EGF-H52H3) for 10 min. After washing with ice-cold PBS, the cells were lysed, and the cell lysates were separated by 4–20% gradient SDS-PAGE under reducing conditions.

Techniques: Western Blot, Concentration Assay

Journal: PLOS One

Article Title: Inhibition of HER2 signaling and breast cancer cell growth with a novel antibody targeting HER2 ECD III/IV

doi: 10.1371/journal.pone.0338127

Figure Lengend Snippet: A-C. Immunoblots showing HER2 downstream signaling in (A) MCF7, (B) BT-474 and (C) SK-BR-3 treated with 10 μg/mL of the indicated antibodies for 24 h, followed by stimulation with 5 nM EGF for 10 min. D-F. Corresponding immunoblots of (D) MCF7, (E) BT-474 and (F) SK-BR-3 cells treated as above but stimulated with 0.5 nM NRG1 for 10 min. The numerical values shown in the figures represent normalized quantitative data.

Article Snippet: For ligand-dependent HER2 signaling, cells were first incubated with 10 μg/mL each antibody or combinations for 24 h, followed by stimulation with 0.5 nM recombinant human NRG1 or 5 nM EGF (Acro Biosystems, EGF-H52H3) for 10 min. After washing with ice-cold PBS, the cells were lysed, and the cell lysates were separated by 4–20% gradient SDS-PAGE under reducing conditions.

Techniques: Western Blot

Journal: PLOS One

Article Title: Inhibition of HER2 signaling and breast cancer cell growth with a novel antibody targeting HER2 ECD III/IV

doi: 10.1371/journal.pone.0338127

Figure Lengend Snippet: A. Cryo-EM map (upper left) depicting the complex composed of three components: HER2 (sky blue), m66 Fab (orange), and trastuzumab Fab (pink). The final model (upper right) incorporating the three components into the map. Superposition of HER2-trastuzumab-pertuzumab structure (PDB: 6OGE) and HER2-m66-trastuzumab structure (lower left). Superposition of HER2-HER3-NRG1 structure (PDB: 7MN5) and HER2-m66-trastuzumab structure (lower right). B. Cryo-EM map (upper left) illustrated the tetrameric complex consisting of four components: HER2 (sky blue), r40 Fab (green), trastuzumab Fab (pink) and pertuzumab Fab (yellow). The final construction (upper right) with the four proteins fitted into the map. Superposition of HER2-trastuzumab-pertuzumab structure (PDB: 6OGE) and HER2-r40-trastuzumab-pertuzumab structure (lower left). Superposition of HER2-HER3-NRG1 structure (PDB: 7MN5) and HER2-r40-trastuzumab-pertuzumab complex (lower right).

Article Snippet: For ligand-dependent HER2 signaling, cells were first incubated with 10 μg/mL each antibody or combinations for 24 h, followed by stimulation with 0.5 nM recombinant human NRG1 or 5 nM EGF (Acro Biosystems, EGF-H52H3) for 10 min. After washing with ice-cold PBS, the cells were lysed, and the cell lysates were separated by 4–20% gradient SDS-PAGE under reducing conditions.

Techniques: Cryo-EM Sample Prep

Journal: Molecular Therapy Oncology

Article Title: Preclinical development and selection of nanobody-based CAR-T cells targeting HER2-positive solid tumors

doi: 10.1016/j.omton.2025.201088

Figure Lengend Snippet: NanoCARs against HER2 are functional and induce activation of a reporter T cell line (A) Structural modeling of the HER2 extracellular domain and binding of selected HER2 nanobodies. (B) Graphical design and representation of nanoCAR constructs. Nanobodies against HER2 were generated in-house and were described before. (C) Evaluation of binding retention of cell-expressed nanoCARs to soluble recombinant HER2 protein, and correlation with Thy1.1 expression using flow cytometry. (D) Graphical representation of a reporter T cell line harboring GFP expression under control of an NFAT-dependent promoter to test nanoCAR-functionality. (E) Quantified increase in green fluorescence of Jurkat reporter T cells upon co-culture with LN229 target cells, as evaluated by IncuCyte live cell imaging. (F) % GFP pos Thy1.1 pos cells after 24 h of co-culture with LN229 cells, as determined by flow cytometry ( n = 1).

Article Snippet: Expression of the HER2-targeting nanoCARs was detected with human Fc-linked recombinant HER2 protein (ACRO Biosystems).

Techniques: Functional Assay, Activation Assay, Binding Assay, Construct, Generated, Recombinant, Expressing, Flow Cytometry, Control, Fluorescence, Co-Culture Assay, Live Cell Imaging

Journal: Molecular Therapy Oncology

Article Title: Preclinical development and selection of nanobody-based CAR-T cells targeting HER2-positive solid tumors

doi: 10.1016/j.omton.2025.201088

Figure Lengend Snippet: Primary nanoCAR-T cells express activation markers and secrete cytokines to a different extent upon recognition of HER2 pos target cells (A) Graphical representation of the co-culture of CAR-T cells and target cells and CAR-mediated induction of T cell activation markers and cytokines. (B) HER2 expression on LN229 glioblastoma, BT474 breast cancer and 624MEL melanoma target cell lines. (C) 4-1BB expression in CAR-T cells upon 12 h of co-culturing with LN229, BT474 or 624MEL cells ( n = 1 donor). (D) Percentages of 4-1BB pos , CD69 pos and CD25 pos CAR-T cells upon 19 h of co-culture with LN229 target cells. Data represent mean ± SD, n = 3 biological repeats. Each dot represents a different donor. (E) Secretion of IFN γ and TNF α by CAR-T cells after 19 h of co-culture with LN229 target cells. Data represent mean ± SD, n = 3 biological repeats. Each dot represents a different donor. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons correction in D and E. Statistical significance is determined for each HER2 nanoCAR condition compared to the 5T2Id control nanoCAR condition.

Article Snippet: Expression of the HER2-targeting nanoCARs was detected with human Fc-linked recombinant HER2 protein (ACRO Biosystems).

Techniques: Activation Assay, Co-Culture Assay, Expressing, Control

Journal: Molecular Therapy Oncology

Article Title: Preclinical development and selection of nanobody-based CAR-T cells targeting HER2-positive solid tumors

doi: 10.1016/j.omton.2025.201088

Figure Lengend Snippet: Selected nanoCAR-T cells kill HER2 pos glioblastoma, breast and melanoma cell lines with varying potency (A) Graphical representation of nanoCAR-T cell-mediated killing of GFP pos HER2 pos 2D tumor models. (B) Live cell images from IncuCyte showing killing of GFP pos 2D tumor models by nanoCAR-T cells, shown for one of three donors tested. (C) Quantification of IncuCyte live cell imaging showing killing of GFP pos target cell lines by nanoCAR-T cells. Data represent mean ± SD, n = 3 biological repeats. (D) Endpoint flow cytometric analysis of absolute target cell counts after co-culture with CAR-T cells for 72 h. Data represent mean ± SD, n = 3 biological repeats. Each dot represents a different donor. (E) Secretion of IFN γ by CAR-T cells after 72 h of co-culture with target cell lines. Data represent mean ± SD, n = 3 biological repeats. Each dot represents a different donor. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001. two-way ANOVA with Dunnett’s multiple comparisons test was used to determine statistical significance in C (significance shown on the graph at 48 h). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons correction in D and E. Statistical significance is determined for each HER2 nanoCAR condition compared to the 5T2Id control nanoCAR condition.

Article Snippet: Expression of the HER2-targeting nanoCARs was detected with human Fc-linked recombinant HER2 protein (ACRO Biosystems).

Techniques: Live Cell Imaging, Co-Culture Assay, Control

Journal: Molecular Therapy Oncology

Article Title: Preclinical development and selection of nanobody-based CAR-T cells targeting HER2-positive solid tumors

doi: 10.1016/j.omton.2025.201088

Figure Lengend Snippet: Lead 1R59b nanoCAR-T cells kill HER2 pos tumor cells in an antigen specific manner (A) Graphical representation of CAR-T cells killing GFP pos HER2 pos target cells but not Katushka2S pos HER2 neg non-target cells. (B) HER2 expression on wild type and HER2-transduced U87 cells. (C) GFP expression in HER2-transduced and Katushka2S expression in wild type U87 cells, before and after transduction for fluorescent markers. (D) Live cell images from IncuCyte showing killing of GFP pos target cells but not Katushka2S pos non-target cells in 2D tumor models by CAR-T cells, shown for one of three donors tested. (E) Quantification of IncuCyte images showing decrease in GFP signal at decreasing effector-to-target ratios (left to right). Data represent mean ± SD, n = 3 biological repeats. (F) Endpoint flow cytometric analysis of % target cell specific killing at decreasing effector-to-target ratios. Data represent mean ± SD, n = 3 biological repeats. (G) Graphical representation of co-culture experiment of HER2 RNA-electroporated U87 cells with lead 1R59b nanoCAR-T cells. (H) HER2 expression at 24 h after electroporation with decreasing amounts of HER2-encoding mRNA, as determined by flow cytometry. (I) Secretion of IFN γ by CAR-T cells after 24 h of co-culturing with HER2 mRNA-electroporated U87 cells. (J) Structural modeling of binding of trastuzumab-derived V H and V L regions to the HER2 extracellular domain. (K) Secretion of IFN γ by CAR-T cells after 96 h of co-culturing with HER2 mRNA-electroporated U87 cells. (L) HER2 expression on human cardiac myocytes. (M) Graphical representation of the co-culture experiment of CAR-T cells and human cardiac myocytes. (N) Endpoint flow cytometric analysis of absolute target cell counts (left), secretion of IFN γ by CAR-T cells (middle), expression of 4-1BB on CAR-T cells (right) after co-culture for 48 h. Data represent mean ± SD, n = 3 biological repeats. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗∗ p ≤ 0.0001. two-way ANOVA with Dunnett’s multiple comparisons test was used to determine statistical significance in E (significance shown on the graph at endpoint). Statistical significance was determined by two-tailed unpaired t tests in I. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons correction in N. Statistical significance is determined for the HER2 CAR conditions compared to the 5T2Id control nanoCAR condition.

Article Snippet: Expression of the HER2-targeting nanoCARs was detected with human Fc-linked recombinant HER2 protein (ACRO Biosystems).

Techniques: Expressing, Transduction, Co-Culture Assay, Electroporation, Flow Cytometry, Binding Assay, Derivative Assay, Two Tailed Test, Control

Journal: BMB Reports

Article Title: Pluripotent stem cell-derived chimeric antigen receptor-natural killer cells targeting epidermal growth factor receptor 2 for cancer immunotherapy

doi: 10.5483/BMBRep.2025-0137

Figure Lengend Snippet: Generation of anti-HER2 CAR-expressing ESCs. (A) Workflow for generating ESC-derived anti-HER2 CAR-NK cells. (B) Representative morphology and fluorescence images of CAR-ESC colonies. Scale bar = 200 μm. (C) Flow cytometry analysis of CAR and GFP expression in CAR-ESCs. ESCs, embryonic stem cells; HER2, human epidermal growth factor receptor 2; CAR, chimeric antigen receptor; scFv, single-chain variable fragment; TM, transmembrane domain; NK, natural killer; WT, wild-type; GFP, green fluorescent protein.

Article Snippet: CAR expression was assessed by staining cells with human HER2 (ACROBiosystems, Newark, DE, USA).

Techniques: Expressing, Derivative Assay, Fluorescence, Flow Cytometry

Journal: BMB Reports

Article Title: Pluripotent stem cell-derived chimeric antigen receptor-natural killer cells targeting epidermal growth factor receptor 2 for cancer immunotherapy

doi: 10.5483/BMBRep.2025-0137

Figure Lengend Snippet: Characterization of anti-HER2 CAR-ESCs. (A) Relative mRNA levels of pluripotency markers (OCT4, NANOG, and SOX2) in CAR-ESCs and WT-ESCs measured by qPCR. Values represent the mean ± SD from three independent biological replicates (n = 3). No significant differences were observed between CAR-ESCs and WT-ESCs. (B) Expression of pluripotency markers (OCT4, SSEA4, and TRA-1-60) in CAR-ESCs was confirmed by immunocytochemistry. Scale bar = 200 μm. (C) G-banding karyotype analysis showing a normal karyotype in CAR-ESCs. Student’s t-tests were used for all statistical comparisons. ESCs, embryonic stem cells; HER2, human epidermal growth factor receptor 2; CAR, chimeric antigen receptor; WT, wild-type; qPCR, quantitative polymerase chain reaction; SD, standard deviation.

Article Snippet: CAR expression was assessed by staining cells with human HER2 (ACROBiosystems, Newark, DE, USA).

Techniques: Expressing, Immunocytochemistry, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: BMB Reports

Article Title: Pluripotent stem cell-derived chimeric antigen receptor-natural killer cells targeting epidermal growth factor receptor 2 for cancer immunotherapy

doi: 10.5483/BMBRep.2025-0137

Figure Lengend Snippet: Generation of NK cells from anti-HER2 CAR-ESCs. (A) Flow cytometric analysis of CD56, CD3, and CD16 expression on CAR-NK cells derived from three CAR-ESC clones after 4 weeks of NK cell differentiation. (B) Flow cytometric analysis showing the expression of CAR and GFP in NK cells derived from the clones. (C) Morphology of ESC-derived HSPCs (2 weeks) and NK cells (3 weeks). Scale bar = 200 μm. (D) Percentages of CD56＋3− and CD56＋16＋ cells in WT-ESC- and CAR-ESC-derived NK cells after 4 weeks of differentiation. Values represent the mean ± SD from three independent biological replicates (n = 3); points represent individual replicates. Student’s t-tests were used for all statistical comparisons. ESCs, embryonic stem cells; HER2, human epidermal growth factor receptor 2; CAR, chimeric antigen receptor; NK, natural killer; WT, wild-type; SD, standard deviation.

Article Snippet: CAR expression was assessed by staining cells with human HER2 (ACROBiosystems, Newark, DE, USA).

Techniques: Expressing, Derivative Assay, Clone Assay, Cell Differentiation, Standard Deviation

Journal: BMB Reports

Article Title: Pluripotent stem cell-derived chimeric antigen receptor-natural killer cells targeting epidermal growth factor receptor 2 for cancer immunotherapy

doi: 10.5483/BMBRep.2025-0137

Figure Lengend Snippet: Phenotypic comparison of anti-HER2 CAR-ESC- and WT-ESC-derived NK cells. Representative flow cytometric analysis showing the expression of (A) activating receptors (NKG2D, NKG2C, NKp30, NKp44, NKp46, DNAM-1, and CD69), (B) inhibitory receptors (KIR2DL1 and KIR2DL2/L3/S2), (C) co-stimulatory receptors (2B4 and FasL), (D) exhaustion markers (LAG-3, TIGIT, and PD-1), and (E) cytolytic granules (granzyme B and perforin) after 4 weeks of differentiation. Values represent the mean ± SD from three independent biological replicates (n = 3). Student’s t-tests were used for all statistical comparisons. ESCs, embryonic stem cells; HER2, human epidermal growth factor receptor 2; CAR, chimeric antigen receptor; NK, natural killer; WT, wild-type; SD, standard deviation.

Article Snippet: CAR expression was assessed by staining cells with human HER2 (ACROBiosystems, Newark, DE, USA).

Techniques: Comparison, Derivative Assay, Expressing, Standard Deviation

Journal: BMB Reports

Article Title: Pluripotent stem cell-derived chimeric antigen receptor-natural killer cells targeting epidermal growth factor receptor 2 for cancer immunotherapy

doi: 10.5483/BMBRep.2025-0137

Figure Lengend Snippet: Cytotoxicity of anti-HER2 CAR-ESC-derived NK cells against HER2＋ and HER2− cancer cells. (A) Histogram plots showing HER2 expression in ovarian cancer cell lines (A2780cis and SKOV3) and breast cancer cell lines (MDA-MB-231 and SKBR3). (B) Quantification of HER2 expression in the four cell lines as normalized mean fluorescence intensity. (C) Cytotoxicity of NK cells derived from three CAR-ESC clones against SKOV3 (HER2＋). Values represent the mean ± SD from three independent biological replicates (n = 3). No significant differences in cytotoxicity were observed among the three clones. (D) Comparison of cytotoxicity between WT-ESC- and CAR-ESC-derived NK cells against HER2＋ and HER2− cancer cell lines. Values represent the mean ± SD from three independent biological replicates (n = 3). Asterisks indicate a significant difference compared to the WT-NK controls. Statistical tests involved one-way analysis of variance for (C) and Student’s t-test for (D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n.s., not significant; ESCs, embryonic stem cells; HER2, human epidermal growth factor receptor 2; CAR, chimeric antigen receptor; NK, natural killer; WT, wild-type; SD, standard deviation.

Article Snippet: CAR expression was assessed by staining cells with human HER2 (ACROBiosystems, Newark, DE, USA).

Techniques: Derivative Assay, Expressing, Fluorescence, Clone Assay, Comparison, Standard Deviation

Journal: Chemical Science

Article Title: Heterodimeric protein entangling motifs: systematic discovery, feature analysis, and topology engineering

doi: 10.1039/d5sc03953c

Figure Lengend Snippet: Design of a bispecific affibody catenane of AffiEGFR and AffiHER2 using the 4X86 motif. (a) SDS-PAGE analysis of l -bsAffi and cat -bsAffi; (b) TEVp-mediated cleavage of cat -bsAffi to prove its catenane topology; (c) binding characterization of cat -bsAffi and l -bsAffi towards EGFR and HER2, respectively.

Article Snippet: Human EGFR and HER2 proteins were purchased from ACROBiosystems as a white powder (100 μg) and dissolved to 8.5 μM in the running buffer (PBST buffer containing 0.1% BSA for titration) as a stock solution for testing.

Techniques: SDS Page, Binding Assay