Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor

doi: 10.1007/s00262-022-03195-4

Figure Lengend Snippet: Cysteines in the hinge facilitate antigen-triggered CAR cluster formation and promote CAR T-cell activity A Gene schematics of CARCys and CARSer targeting HER2. B Human T cells derived from healthy donors were transduced with or without HER2-CARCys or HER2-CARSer mRNA to generate NT or HER2-CAR-T cells. Twelve hours post-transduction, the CAR expression on these T cells was detected by flow cytometry. Data were representative of at least three independent experiments. C–E CARCys and CARSer distribution on these T cells was observed using STORM system after stimulation with HER2 antigen for 30 min. The tables containing x × y co-ordinates of each CAR molecule were obtained from STORM, and DBSCAN implemented in MATLAB was used for CAR clusters identification. For each T cell, a 5 × 5 μm2 ROI in cell center was selected for further analysis of cluster number and diameter by DBSCAN. C The representative cluster maps of CARCys and CARser on primary human T cells. D The number of CAR clusters per ROI. E The diameters of CAR clusters. Data shown as mean ± SEM were from 13 to 16 cells of healthy donors. F CARCys and CARSer-T cells were co-cultured with HER2+N87 or HER2−K562 tumor cells. Four hours later, CD107a translocation of CAR-T cells was detected by flow cytometry. Data shown as mean ± SEM were pooled from three healthy donors. G, H CARCys and CARSer-T cells were co-cultured with HER2+N87 at indicated E: T ratios for 6 or 48 h. The cytotoxicity of CAR-T cells was calculated by the target cells’ viability using CCK8. Data shown as mean ± SEM were from one of two independent experiments using two healthy donors. P values were determined by unpaired Student’s t test in (D, E), and two-way ANOVA in F, G, and H. **P < 0.01, ***P < 0.001

Article Snippet: CAR-T cells were stimulated with 2 μg/mL recombinant human HER2 protein (10,004-H02H-B, Sino Biological Inc) for 30 min at 37 °C.

Techniques: Activity Assay, Derivative Assay, Transduction, Expressing, Flow Cytometry, Cell Culture, Translocation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor

doi: 10.1007/s00262-022-03195-4

Figure Lengend Snippet: CARCys-T cells exhibit increased antigen-specific activity compared to CARconv-T cells in vitro Human T cells derived from healthy donors were transduced with or without HER2-CARconv or HER2-CARCys mRNA to generate NT or HER2-CAR-T cells. A These CAR-T cells were stimulated with HER2 antigen for 30 min, and identified the antigen-triggered CAR clusters by STROM and DBSCAN analysis. B, C The number of CAR clusters per ROI (5 × 5 mm2) and the diameter of CAR clusters were calculated by DBSCAN analysis. Data shown as mean ± SEM were from 10 CAR-T cells of healthy donors. D Twelve hours post-transduction, NT- or CAR-T cells were cultured in Opti-MEM for 6 h to reduce background. These T cells were incubated with biotinylated HER2 protein (2.5 μg/mL) for 30 min at 4 °C and washed twice with cold PBS. Then, these T cells were cross-linked with streptavidin (5 μg/mL) at 37 °C for 30 s to measure the phosphorylation of CD3ζ and ZAP70, and cross-linked for 2 min to detect the phosphorylation of ERK1/2, S6 and AKT. E The enriched gene sets of CARconv-T cell comparing with CARCys-T cells after 10-h antigen stimulation. F The heat map of selected differentially expressed genes related to T cell activation, cytokine production, cytotoxicity, and chemokine signaling. Differentially expressed genes were selected with an adjusted P value (false discovery rate, FDR) < 0.05 and absolute fold change ≥ 2. G–J NT, CARconv, or CARCys-T cells were co-cultured with HER2−K562 or HER2+N87. (G) CD25 and Ki67 expressions of T cells were detected by flow cytometry 24 and 48 h post-co-culture, respectively. Data shown as mean ± SEM were pooled from four healthy donors. H, I IFN-γ and IL-2 production of T cells were measured by ELISA 24 h post-co-culture. Data shown as mean ± SEM were from one of three independent experiments using three healthy donors. J Cytotoxic activity of T cells was measured by performing CCK8 assay 12 h post-co-culture at indicated E: T ratios; data shown as mean ± SEM were from one of two independent experiments using two healthy donors. K–M Human T cells were transduced with HER2-CARconv, HER2-CARCys or HER2-CARSer mRNA to generate CAR-T cells. Twelve hours post-transduction, these CAR-T cells were treated with 10 mM/mL DTT and co-cultured with HER2+N87 tumor cells at the ratio of 1: 2. Six hours later, the CD107a translocation (K), the TNF-α (L) and IFN-γ (M) production were detected by flow cytometry. Data shown as mean ± SEM were from two independent experiments. P values were determined by unpaired Student’s t-test in (C), one-way ANOVA in (G), and two-way ANOVA in (H–M). *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: CAR-T cells were stimulated with 2 μg/mL recombinant human HER2 protein (10,004-H02H-B, Sino Biological Inc) for 30 min at 37 °C.

Techniques: Activity Assay, In Vitro, Derivative Assay, Transduction, Cell Culture, Incubation, Activation Assay, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Translocation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor

doi: 10.1007/s00262-022-03195-4

Figure Lengend Snippet: CARCys-T cells with different CAR framework exhibit stronger antigen-specific activity than CARconv-T cells. Human T cells derived from healthy donors were transduced with or without second-generation HER2-CARconv or HER2-CARCys with a 4-1BB costimulatory domain, or third-generation CD19-CARconv or CD19-CARCys mRNA to generate NT- or CAR-T cells. A Twelve hours post-transduction, HER2-CAR expression levels of NT-, HER2-CARconv-, or HER2-CARCys-T cells were detected by flow cytometry. B NT-, HER2-CARconv- or HER2-CARCys-T cells were co-cultured with HER2−K562 or HER2+N87. Twenty-four hours post-co-culture, IFN-γ production of these T cells were measured by ELISA. Data shown as mean ± SEM were from one of two independent experiments using two healthy donors. C CD107a translocation of NT-, HER2-CARconv-, or HER2-CARCys-T cells was detected by flow cytometry four hours post-co-culture. D Twelve hours post-co-culture, the cytotoxicity of NT-, HER2-CARconv-, or HER2-CARCys-T cells was analyzed by performing CCK8 assay after at indicated E: T ratios. Data shown as mean ± SEM were from one of two independent experiments using two healthy donors. E Twelve hours post-transduction, CD19-CAR expression levels of NT-, CD19-CARconv-, or CD19-CARCys-T cells were detected by flow cytometry. F, G NT-, CD19-CARconv-, or CD19-CARCys-T cells were co-cultured with CD19+Ramos for 12 h. CD25, CD69, CD95, and Perforin expression levels and cytokine production (TNF-α, IFN-γ) of these T cells were detected by flow cytometry. H NT-, CD19-CARconv-, or CD19-CARCys-T cells were co-cultured with CD19− K562 or CD19+ Ramos. CD107a translocation of NT, CD19-CARconv, or CD19-CARCys-T cells was detected by flow cytometry four hours post-co-culture. Data shown as mean ± SEM were pooled from two healthy donors. P values were determined by two-way ANOVA in (B, D, H). *P < 0.05, and **P < 0.01

Article Snippet: CAR-T cells were stimulated with 2 μg/mL recombinant human HER2 protein (10,004-H02H-B, Sino Biological Inc) for 30 min at 37 °C.

Techniques: Activity Assay, Derivative Assay, Transduction, Expressing, Flow Cytometry, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Translocation Assay, CCK-8 Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Inhibition of growth and HER2 signaling by HER2Ab-NSCs cells. (A) Inhibition in growth of BT474 cells using supernatant of HER2Ab-NSCs. (B) Co-Culture assay using BT474 cells with either vector control or HER2Ab-NSCs cells. (C-D) Inhibition of PI3K-AKT signaling using purified anti-HER2Ab released by NSC. Right panel in C and D demonstrates significant decrease in relative densitometric units. Trastuzumab was used as positive control in A, C and D. The experiments were repeated three times. * indicates p<0.05 and ** indicates p<0.01.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Co-culture Assay, Plasmid Preparation, Control, Purification, Positive Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

doi: 10.1002/stem.2109

Figure Lengend Snippet: Binding of anti-HER2Ab to HER2 overexpressing breast cancer cells. (A) BT474Br were stained with trastuzumab as a positive control (B-F) MCF 7, MDA-MB-361, BT474Br, ZR-75-30 and SKBR3 cells stained with anti-HER2Ab secreted by NSC. The experiments were repeated two times.

Article Snippet: ELISA plates (Corning, Pittsburg, PA) were coated overnight with 2μg/mL of recombinant human HER2 Fc chimera (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Staining, Positive Control

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Gating strategy for detection of hHER2-CAR-T cells. A median of > 90% of mouse CD8 T cells transduced with the hHER2-CAR retrovirus were positive for the transgene as judged by flow cytometry analysis. B. B16F10 cells were transfected with mammalian human HER2 construct with lipofectamine. Cells were grown in the presence of G418 and sorted twice to generate single cell clones. C. CAR-T-mediated B16-HER2 cell killing assay. Apoptotic cells were stained for Annexin V and analyzed by flow cytometry. D. Whole mouse lung before (left) and after (right) CUBIC clearing.

Article Snippet: Biotinylated goat anti-rat IgG (H+L) was purchased from Vector Lab. Recombinant human ErbB2/Her2 Fc chimera protein and human ErbB2/Her2-Alexa Fluor-594 were obtained from R&D systems.

Techniques: In Vivo, Transduction, Flow Cytometry, Transfection, Construct, Clone Assay, Staining

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Generation of GFP-ROSAβII-spectrin mouse model (Rosa26tm(CAG-LSL-Sptbn1-IRES-GFP)). Mouse Sptbn1 cDNA was inserted into the CAG-STOP-GFP-Rosa targeting vector, CTV, between a floxed Stop cassette and the internal ribosome entry site (IRES) followed by the enhanced Green Fluorescent Protein gene (eGFP). Transcription is under control of the CAG promoter. The targeting vector contained Rosa26 homology arms (1 kb 5′ and 3.8 kb 3′), so that the entire loxP-stop-loxP-Tmc2-IRES-GFP transcriptional cassette was inserted into the first intron of Rosa26 gene on chromosome 6. B & C. Flow cytometry analysis of IFNγ and TNFα expression in hHER2-CAR transfected T cells from GFP-ROSAβII-spectrin mouse (TβII-spectrin) after treated with PBS or Tat-Cre Recombinase. Cells were co-cultured with B16-HER2 cells. Data represent mean ± SEM. n = 9. D. CAR-mediated B16-HER2 cell killing assay with TβII-spectrin cells after treated with PBS or Tat-Cre Recombinase. Cell death was stained for NucSpot. Data represent mean ± SEM. n = 9. E. Activated CD4+ T cell (OT-II) migration on ICAM-1 coated plates ± CXCL12. Cells were treated with PTx (6 hr) or Gallein (30 min) where indicated. Data were collected from 2 independent experiments (n = 2, 17–34 individual cells per mouse). Data represent mean ± SEM Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. *P = 0.007. F. The pie charts depict the proportion of CD4 T cells distributed in the tumour, blood, LN/spleen, or lung/liver 72 h post-injection. G. Expression levels of βII-spectrin and St3gal1 in human CD4 and CD8 T cells (before and after activation). Loading control: β-actin. Representative western blot images from three independent experiments are shown. H. Expression levels of βII-spectrin in human CD8 memory T cells (CD8+CD45RO+CD45RA–CD56–CD57–). Loading control: β-actin. Representative western blot images from three independent experiments are shown.

Article Snippet: Biotinylated goat anti-rat IgG (H+L) was purchased from Vector Lab. Recombinant human ErbB2/Her2 Fc chimera protein and human ErbB2/Her2-Alexa Fluor-594 were obtained from R&D systems.

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, Transfection, Cell Culture, Staining, Migration, Injection, Activation Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

doi: 10.3390/ijms18081688

Figure Lengend Snippet: Scheme of the Z HER2:2891 -Fc-MMAE homodimer.

Article Snippet: The reduced Z HER2:2891 -Fc was incubated with a 16 molar excess of MC-Val-Cit-PABC-MMAE (maleimidocaprylvaline-citruline- p -amino-benzyloxycarbonyl-mono-methylauristatin E) (ChiroBlock, Wolfen, Germany) for 3 h with gentle mixing at 4 °C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

doi: 10.3390/ijms18081688

Figure Lengend Snippet: Expression and purification of Z HER2:2891 -Fc. ( a ) The levels of Z HER2:2891 -Fc in the CHO-S cells culture medium; ( b ) Western blot analysis of the purification of Z HER2:2891 -Fc; ( c ) The mass spectrum of the purified Z HER2:2891 -Fc.

Article Snippet: The reduced Z HER2:2891 -Fc was incubated with a 16 molar excess of MC-Val-Cit-PABC-MMAE (maleimidocaprylvaline-citruline- p -amino-benzyloxycarbonyl-mono-methylauristatin E) (ChiroBlock, Wolfen, Germany) for 3 h with gentle mixing at 4 °C.

Techniques: Expressing, Purification, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

doi: 10.3390/ijms18081688

Figure Lengend Snippet: Specificity of the Z HER2:2891 -Fc binding. ( a ) Fluorescent microscopy images of the HER2-positive SK-BR-3 and MDA-MB-231 HER2-negative cells. The anti-HER2 antibody was detected with a FITC-labeled secondary antibody whereas the affibody was FITC labelled, scale bar = 100 µm; ( b ) Z HER2:2891 -Fc binding to the SK-BR-3 and MDA-MB-231 cells analyzed by Western blotting. The upper panel shows HER2 levels in SK-BR-3 and MDA-MB-231cells, the middle panel severs as a loading control for the experiment, the bottom panel demonstrates that Z HER2:2891 -Fc is specifically enriched at the surface of the SK-BR-3 cells.

Article Snippet: The reduced Z HER2:2891 -Fc was incubated with a 16 molar excess of MC-Val-Cit-PABC-MMAE (maleimidocaprylvaline-citruline- p -amino-benzyloxycarbonyl-mono-methylauristatin E) (ChiroBlock, Wolfen, Germany) for 3 h with gentle mixing at 4 °C.

Techniques: Binding Assay, Microscopy, Labeling, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

doi: 10.3390/ijms18081688

Figure Lengend Snippet: Conjugation of MMAE to Z HER2:2891 -Fc. ( a ) The Z HER2:2891 -Fc species before and after conjugation separated on 12% SDS-PAGE gel and subjected to Coomassie staining. The samples were heated (96 °C, 10 min) in non-reducing Laemmli buffer prior the electrophoretic separation; ( b ) The mass spectrum of the conjugation product; ( c ) The Z HER2:2891 -Fc-MMAE conjugate migration on a native 12% polyacrylamide gel under non-reducing conditions stained with Coomassie .

Article Snippet: The reduced Z HER2:2891 -Fc was incubated with a 16 molar excess of MC-Val-Cit-PABC-MMAE (maleimidocaprylvaline-citruline- p -amino-benzyloxycarbonyl-mono-methylauristatin E) (ChiroBlock, Wolfen, Germany) for 3 h with gentle mixing at 4 °C.

Techniques: Conjugation Assay, SDS Page, Staining, Migration

Journal: International Journal of Molecular Sciences

Article Title: Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

doi: 10.3390/ijms18081688

Figure Lengend Snippet: Cytotoxicity of the Z HER2:2891 -Fc-MMAE conjugate. The conjugate was tested on four breast cancer cell lines: ( a ) SK-BR-3 (HER2+); ( b ) MDA-MB-453 (HER2+); ( c ) T-47-D (HER2-) and ( d ) MDA-MB-231 (HER2-). Error bars represent standard error of the mean (SEM).

Article Snippet: The reduced Z HER2:2891 -Fc was incubated with a 16 molar excess of MC-Val-Cit-PABC-MMAE (maleimidocaprylvaline-citruline- p -amino-benzyloxycarbonyl-mono-methylauristatin E) (ChiroBlock, Wolfen, Germany) for 3 h with gentle mixing at 4 °C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

doi: 10.3390/ijms18081688

Figure Lengend Snippet: The cytotoxicity of Z HER2:2891 -Fc-MMAE in comparison to the previously published IC 50 values obtained for the Z HER2:2891 -MMAE conjugate. IC 50 results are expressed as mean ± SEM.

Article Snippet: The reduced Z HER2:2891 -Fc was incubated with a 16 molar excess of MC-Val-Cit-PABC-MMAE (maleimidocaprylvaline-citruline- p -amino-benzyloxycarbonyl-mono-methylauristatin E) (ChiroBlock, Wolfen, Germany) for 3 h with gentle mixing at 4 °C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

doi: 10.3390/ijms18081688

Figure Lengend Snippet: The stability of Z HER2:2891 -Fc-MMAE in ( a ) human and ( b ) mouse plasma analyzed by Western blotting. The asterisk indicates a nonspecific band.

Article Snippet: The reduced Z HER2:2891 -Fc was incubated with a 16 molar excess of MC-Val-Cit-PABC-MMAE (maleimidocaprylvaline-citruline- p -amino-benzyloxycarbonyl-mono-methylauristatin E) (ChiroBlock, Wolfen, Germany) for 3 h with gentle mixing at 4 °C.

Techniques: Western Blot

Journal: ACS Central Science

Article Title: Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs)

doi: 10.1021/acscentsci.2c01437

Figure Lengend Snippet: Chemical construction of a full antibody to generate Fc CD20 -(Fab HER2 ) 2 SynAb 1. A Mono-PD method for the construction of a full antibody Fc CD20 -(Fab HER2 ) 2 SynAb 1 . Fc CD20 5 is sequentially reduced and rebridged with Br 2 PD-BCN 4 . The resulting Fc CD20 -(BCN) 2 6 is then reacted with Fab HER2 -Tz 7 to generate Fc CD20 -(Fab HER2 ) 2 SynAb 1 after SEC purification. B Br 2 PD-Tz 3 used for SynAb synthesis. C Br 2 PD-BCN 4 used for SynAb synthesis. D SDS-PAGE analysis of Fc CD20 -(Fab HER2 ) 2 SynAb 1 formation. Lane 1: Ladder. Lane 2: Fab HER2 8 . Lane 3: Fc CD20 5 . Lane 4: Crude Fc CD20 -(Fab HER2 ) 2 SynAb 1 . E UV trace of SEC purification of Fc CD20 -(Fab HER2 ) 2 SynAb 1 . F SDS-PAGE analysis of SEC purification of SynAb 1 . Lane 1: Ladder. Lane 2–3: Aggregates. Lane 4–6: SynAb 1 . Lane 7–8: BCN-Fc CD20 -Fab HER2 . Lane 11–14: Fab HER2 8 . G LC-MS analysis of Fc CD20 5 . Observed mass: 49868 Da. H LC-MS analysis of Fab HER2 -Tz 7 . Expected mass: 48334 Da. Observed mass: 48321 Da. I , J LC-MS analysis of Fc CD20 -(BCN) 2 6 . Expected mass: 50873 Da (Fc CD20 -(BCN) 2 6 ) and 25438 Da (disulfide scrambled Fc CD20 -(BCN) 2 6 ). Observed mass: 50874 and 25437 Da. K , L LC-MS analysis of Fc CD20 -(Fab HER2 ) 2 SynAb 1 . Expected mass: 147486 Da (Fc CD20 -(Fab HER2 ) 2 1 ) and 73744 Da (disulfide scrambled Fc CD20 -(Fab HER2 ) 2 1 ). Observed mass: 147491 Da, 148246 Da (Δ = 755 Da) and 73745 Da.

Article Snippet: The production of the Fc CD20 -(Fab HER2 ) 2 SynAbs 1 and 2 was successful.

Techniques: Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: ACS Central Science

Article Title: Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs)

doi: 10.1021/acscentsci.2c01437

Figure Lengend Snippet: Chemical construction of a full antibody to generate Fc CD20 -(Fab HER2 ) 2 SynAb 2. A Bis-PD method for the construction of a full antibody Fc CD20 -(Fab HER2 ) 2 SynAb 2 . Fc CD20 5 is sequentially reduced and rebridged with bis-PD(Tz) 2 9 . The resulting Fc CD20 -(Tz) 2 10 is then reacted with Fab HER2 -BCN 11 to generate Fc CD20 -(Fab HER2 ) 2 SynAb 2 after SEC purification. B Bis-PD(Tz) 2 9 used for SynAb synthesis. C Br 2 PD-BCN 4 used for SynAb synthesis. D SDS-PAGE analysis of Fc CD20 -(Fab HER2 ) 2 SynAb 2 formation. Lane 1: Ladder. Lane 2: Fab HER2 -BCN 11 . Lane 3: Fc CD20 -(Tz) 2 10 . Lane 4: crude Fc CD20 -(Fab HER2 ) 2 SynAb 2 . E UV trace of SEC purification of Fc CD20 -(Fab HER2 ) 2 SynAb 2 . F SDS-PAGE analysis of SEC purification of Fc CD20 -(Fab HER2 ) 2 SynAb 2 . Lane 1: Ladder. Lane 2–5: Large species. Lane 6–9: Fc CD20 -(Fab HER2 ) 2 SynAb 2 . Lane 10–12: Fc CD20 -(Fab HER2 )-Tz 12 . G LC-MS analysis of Fc CD20 -(Tz) 2 10 . Expected mass: 51246 Da. Observed mass: 51250 Da. H LC-MS analysis of Fab HER2 -BCN 11 . Expected mass: 48141 Da. Observed mass: 48141 Da. I LC-MS analysis of Fc CD20 -(Fab HER2 )-Tz 12 . Expected mass: 99359 Da. Observed mass: 99367 Da. J LC-MS analysis of Fc CD20 -(Fab HER2 ) 2 SynAb 2 . Expected mass: 147472 Da. Observed mass: 147488 Da.

Article Snippet: The production of the Fc CD20 -(Fab HER2 ) 2 SynAbs 1 and 2 was successful.

Techniques: Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: ACS Central Science

Article Title: Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs)

doi: 10.1021/acscentsci.2c01437

Figure Lengend Snippet: Chemical generation of bispecific Fc CD20 -(Fab HER2 )-Fab CD3 SynAb 13 . A Bis-PD method for the construction of bispecific Fc CD20 -(Fab HER2 )-Fab CD3 SynAb 13 . Fc CD20 5 is sequentially reduced and rebridged with bis-PD(Tz) 2 9 . The resulting Fc CD20 -(Tz) 2 10 is then reacted with Fab HER2 -BCN 11 to generate Fc CD20 -(Fab HER2 )-Tz 12 after SEC purification. Fc CD20 -(Fab HER2 )-Tz 12 is reacted further with Fab CD3 -BCN 15 to generate bispecific Fc CD20 -(Fab HER2 )-Fab CD3 SynAb 13 after SEC purification. B The bis-PD(Tz) 2 9 used for bispecific SynAb synthesis. C Br 2 PD-BCN 4 used for bispecific SynAb synthesis. D UV trace of SEC purification of Fc CD20 -(Fab HER2 )-Tz 12 . E SDS-PAGE analysis of Fc CD20 -(Fab HER2 )-Tz 12 intermediate formation and SEC purification. Lane 1: Ladder. Lane 2: Anti-HER2 mAb (trastuzumab). Lane 3: Fc CD20 -(Tz) 2 10 . Lane 4: Fab HER2 -BCN 11 . Lane 5: Crude Fc CD20 -(Fab HER2 )-Tz 12 . Lane 6–7 and 13: Fc CD20 -(Fab HER2 ) 2 2 byproduct/larger species. Lane 8–9: Purified Fc CD20 -(Fab HER2 )-Tz 12 . Lane 10–12: Left-over Fab and Fc species. F LC-MS analysis of Fc CD20 5 . Observed mass: 49868 Da. G LC-MS analysis of Fc CD20 -(Tz) 2 10 . Expected mass: 51246 Da. Observed mass: 51250 Da. H LC-MS analysis of Fab HER2 -BCN 11 . Expected mass: 48141 Da. Observed mass: 48141 Da. I LC-MS analysis of Fab CD3 -BCN 15 . Expected mass: 47948 Da. Observed mass: 47950 Da. J LC-MS analysis of Fc CD20 -(Fab HER2 )-Tz 12 . Expected mass: 99359 Da. Observed mass: 99367 Da. K UV trace of SEC purification of bispecific Fc CD20 -(Fab HER2 )-Fab CD3 SynAb 13 . L SDS-PAGE analysis of SEC purification of Fc CD20 -(Fab HER2 )-Fab CD3 SynAb 13 . Lane 1: Ladder. Lane 2: Anti-HER2 mAb (trastuzumab). Lane 3: Fc CD20 -(Tz) 2 10 . Lane 4: Fab HER2 -BCN 11 . Lane 5: Fc CD20 -(Fab HER2 )-Tz 12 intermediate. Lane 6–8: Large species. Lane 9–10: Fc CD20 -(Fab HER2 )-Fab CD3 SynAb 13 . Lane 11–12: Left-over Fc CD20 -(Fab HER2 )-Tz 12 . M LC-MS analysis of bispecific Fc CD20 -(Fab HER2 )-Fab CD3 SynAb 13 . Expected mass: 147284 and 147398 Da. Observed mass: 147292 and 147396 Da.

Article Snippet: The production of the Fc CD20 -(Fab HER2 ) 2 SynAbs 1 and 2 was successful.

Techniques: Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: Dual membrane receptor degradation via folate receptor targeting chimera

doi: 10.1038/s41467-025-63882-5

Figure Lengend Snippet: a A schematic illustration of FolTAC-mediated endocytosis of two POIs. FolTAC induces proximity of FRα and a POI at the cell surface, leading to co-internalization of the complex to the endosome/lysosome for degradation. b Schematics and structures of the knob-into-hole and the string FolTAC-dual geometry. c Relative cell surface FRα expression levels across various non-tumorigenic and cancer cell lines characterized by flow cytometry. Cell lines were grouped into five categories: normal (black downward-pointing triangles), gynecologic cancer (orange squares), breast cancer (olive-green upward-pointing triangles), lung cancer (green circles), and other cancers (red stars). Normal cells (HEK293T, HFF-1, IMR-90, h-8LD, Primary B cells, and Primary T cells) served as the control group for statistical comparisons. Each data point represents the mean fluorescence intensity (MFI) of FRα staining. Data are presented as mean ± standard deviation (SD), with n = 3 biological replicates per condition. The quantification of the data was analyzed using two-sided Student’s t -tests using GraphPad Prism software, and exact p -values are shown above the figure bars. Differences were considered statistically significant at p < 0.05. *, ***, ****: p < 0.05, p < 0.001, p < 0.0001. d Western blotting of HER2 degradation by HER2 Trastuzumab (Traz) FolTAC v0.1, HER2 affibody without Cathepsin B linker FolTAC v0.2, and HER2 affibody with Cathepsin B linker FolTAC v0.3 in SKBR3 cells. Relative protein levels were labeled with numbers below the indicated bands. e Western blotting of EGFR degradation by various EGFR isotypes v0.2, v0.4, and v0.5 in A549 cells. Relative protein levels were labeled with numbers below the indicated bands. f Schematics of the evolution of FolTAC by rational design, including the ligand, linker, and isotype optimization. g Maximal degradation efficiency ( D max , %) of FolTAC across various targets achieved through rational design. Created in BioRender. WANG, Z. (2025) https://BioRender.com/d5n9vj6 .

Article Snippet: Binding affinity measurements of HER2 affibody-Fc and Trastuzumab in both SKBR3-WT and Traz/Lap-resistant SKBR3 cells revealed no significant difference (Supplementary Fig. ), suggesting that the enhanced efficacy of FolTAC-dual may arise from its cooperative configuration or multivalent engagement, rather than from intrinsic differences in binding strength.

Techniques: Expressing, Flow Cytometry, Control, Fluorescence, Staining, Standard Deviation, Software, Western Blot, Labeling

Journal: Nature Communications

Article Title: Dual membrane receptor degradation via folate receptor targeting chimera

doi: 10.1038/s41467-025-63882-5

Figure Lengend Snippet: a Schematic illustrating the geometry-based evolution of FolTAC-dual. b EGFR/HER2 FolTAC-dual designs: knob-into-hole v0.6 and string v1.0 formats. c – e Western blot analysis of EGFR and HER2 degradation in SKBR3 cells by EGFR/HER2 FolTAC-dual, comparing knob-into-hole v0.6 ( c ) and string v1.0 ( d ), with quantification shown in ( e ). The EGFR/HER2 bispecific antibody lacking the FRα scFv served as the control. f Design schematics of PD-L1/VISTA FolTAC-dual in knob-into-hole v0.6 and string v1.0 formats. g – i Western blot analysis of PD-L1 and VISTA degradation in SUM159PT cells by PD-L1/VISTA FolTAC-dual, comparing knob-into-hole v0.6 ( g ) and string v1.0 ( h ), with quantification shown in ( i ). The control construct matched v1.0 but lacked the Farletuzumab arm. Samples and blots for Fig. 2e, i came from the same experiment and were processed in parallel. j Schematic of the competition binding assay to assess the relative binding affinity of knob-into-hole v0.6 and string v1.0. k EGFR receptor levels detected by flow cytometry using a V5-647 antibody after treatment with increasing concentrations of EGFR affibody competitor. The analysis compared the internalization efficiency of the knob-into-hole (magenta squares) and string (cyan circles) formats. A conventional, untreated, or vehicle-only control group was not included, as the experiment was designed for direct comparison between the two formats. l IC₅₀ determination for EGFR engagement by knob-into-hole (red curve) and string (blue curve) formats. m Western blot analysis of EGFR levels following competition with affibody-Fc to evaluate degradation by knob-into-hole and string constructs. No untreated control was included to enable direct head-to-head comparison. n Schematic of geometry-influenced EGFR binding, with dissociation constants ( K D ) calculated using the Cheng–Prusoff equation. Data in panels ( e ), ( i ), ( k ), ( l ), and ( m ) represent three independent biological replicates ( n = 3) and are presented as mean ± SD. Statistical significance in panels 2e and 2i was assessed using two-sided Student’s t -tests, and exact p -values are shown above the figure bars. p < 0.05 was considered significant. p values: * <0.05, ** <0.01, *** <0.001, **** <0.0001. Created in BioRender. WANG, Z. (2025) https://BioRender.com/d5n9vj6 .

Article Snippet: Binding affinity measurements of HER2 affibody-Fc and Trastuzumab in both SKBR3-WT and Traz/Lap-resistant SKBR3 cells revealed no significant difference (Supplementary Fig. ), suggesting that the enhanced efficacy of FolTAC-dual may arise from its cooperative configuration or multivalent engagement, rather than from intrinsic differences in binding strength.

Techniques: Western Blot, Control, Construct, Binding Assay, Flow Cytometry, Comparison

Journal: Nature Communications

Article Title: Dual membrane receptor degradation via folate receptor targeting chimera

doi: 10.1038/s41467-025-63882-5

Figure Lengend Snippet: a Schematic illustration of Red-Farletuzumab pHrodo to analyze endocytosis comparing cancer versus normal cells. pHrodo is a fluorescent dye with minimal fluorescence at neutral pH but exhibits strong fluorescence in acidic environments following internalization. Both cancer and normal non-tumorigenic cells were incubated with pHrodo Red-Farletuzumab for four hours, and the internalization was assessed using flow cytometry. b Flow cytometry analysis of FRα endocytosis. Cancer cells demonstrated a significantly higher capacity to internalize the pHrodo Red-Farletuzumab conjugates compared to normal non-tumorigenic cells. Data are presented as mean ± SD ( n = 3). The P value was calculated using an unpaired two-sided Student’s t-test. c Schematics of the strategy to understand the trafficking of CAR-GFP mediated by FolTAC and co-localization imaging of CAR-GFP with lysosomal marker-Lamp1b-mCherry. Cells were treated with PBS, Control, or CAR FolTAC at 100 nM for 24 h before performing the imaging. CAR-GFP (green) marks CAR-expressing cells, Lamp1b-mCherry (magenta) labels lysosomes, and Hoechst 33342 (blue) stains nuclei. Merged images highlight subcellular localization and co-localization patterns. Scale bar: 20 μm (white). d Study of the degradation pathway of FolTAC in PC9 cells using the lysosome inhibitor chloroquine (CQ), bafilomycin (Baf), and the proteasome inhibitor MG132. e Competitive rescue of HER2 FolTAC-mediated degradation using FC-Farletuzumab in SKBR3 cells. f Flow cytometry analysis of FOLR1 knockdown SUMP159PT cells. g Western blot analysis of PD-L1 degradation following treatment with PD-L1 FolTAC in SUM159PT FOLR1 knockdown and WT cells. Untreated (0 nM) samples served as the control group for comparison. Quantification analysis of PD-L1 level in SUM159PT FOLR1 knockdown cells treated with or without FolTAC. “UT” denotes untreated controls. Data represent three independent biological replicates and are presented as mean ± SD. Statistical analysis was performed using an unpaired two-sided Student’s t-test, and exact p -values are shown above the figure bars. h Flow cytometry analysis of FRα overexpression in MC38 cells. i , j mPD-L1 degradation mediated by mPD-L1 control ( i ) in MC38, and FolTAC in MC38-WT and MC38- hFOLR1 cells ( j ). k Western blot analysis of VISTA and FRα co-degradation in MC38 cells overexpressed with FRα and VISTA. Created in BioRender. WANG, Z. (2025) https://BioRender.com/d5n9vj6 .

Article Snippet: Binding affinity measurements of HER2 affibody-Fc and Trastuzumab in both SKBR3-WT and Traz/Lap-resistant SKBR3 cells revealed no significant difference (Supplementary Fig. ), suggesting that the enhanced efficacy of FolTAC-dual may arise from its cooperative configuration or multivalent engagement, rather than from intrinsic differences in binding strength.

Techniques: Fluorescence, Incubation, Flow Cytometry, Imaging, Marker, Control, Expressing, Knockdown, Western Blot, Comparison, Over Expression

Journal: Nature Communications

Article Title: Dual membrane receptor degradation via folate receptor targeting chimera

doi: 10.1038/s41467-025-63882-5

Figure Lengend Snippet: a Schematic overview of FolTAC-dual therapy for HER2⁺ breast cancer. Left: therapeutic progression in HER2⁺ BC from conventional antibody therapy to dual-targeted degradation. Right: ternary complex formed by FolTAC-dual engaging EGFR, HER2, and FRα to enable selective endocytosis and lysosomal degradation. b Cell viability curves of SKBR3-WT and Traz/Lap-resistant SKBR3 cells treated with Trastuzumab, Lapatinib, or Combo (PC, Carbo/Taxol combination). WT cells served as the reference group for comparative profiling against the resistant line. c Cell viability of normal human cell lines (16HBE, BEAS-2B, IMR-90, LF-1, HFF-1) and cancer cells (SKBR3-WT and Traz/Lap-resistant SKBR3) following treatment with Combo or EGFR/HER2 FolTAC-dual v1.0. d – e Co-culture assays evaluating selective anti-cancer activity of FolTAC-dual. SKBR3-WT ( d ) or Traz/Lap-resistant SKBR3 ( e ) cells expressing GFP were co-cultured with HFF-1 fibroblasts expressing mCherry and treated with Trastuzumab, Combo, or EGFR/HER2 FolTAC-dual v1.0 (100 nM for 6 days). SKBR3 cells are shown in green and HFF-1 fibroblasts in magenta. Cell morphology and spatial distribution were monitored by fluorescence microscopy. Scale bar: 200 μm (white). f–h Quantification of mean fluorescence intensity (MFI) in co-cultures shown in ( d – e ). f MFI of SKBR3-WT-GFP and HFF-1-mCherry from ( d ). ( g ) MFI of Traz/Lap-resistant SKBR3-GFP and HFF-1-mCherry from ( e ). h MFI of HFF-1-mCherry from both experiments, assessing off-target toxicity. “UT” denotes untreated control samples. For ( b – c , f – h ), each data point represents the mean ± SD from three independent biological replicates ( n = 3), each corresponding to a separately seeded and treated well of cells from distinct passages. No technical replicates were used. The unit of study was one well of cell culture. MFIs were quantified using ImageJ software. Statistical comparisons were performed using two-sided unpaired Student’s t -tests in GraphPad Prism. No corrections for multiple comparisons were applied. Exact p -values are shown above bars. Differences were considered statistically significant at p < 0.05. Significance annotations: ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Created in BioRender. WANG, Z. (2025) https://BioRender.com/d5n9vj6 .

Article Snippet: Binding affinity measurements of HER2 affibody-Fc and Trastuzumab in both SKBR3-WT and Traz/Lap-resistant SKBR3 cells revealed no significant difference (Supplementary Fig. ), suggesting that the enhanced efficacy of FolTAC-dual may arise from its cooperative configuration or multivalent engagement, rather than from intrinsic differences in binding strength.

Techniques: Co-Culture Assay, Activity Assay, Expressing, Cell Culture, Fluorescence, Microscopy, Control, Software

Journal: Nature Communications

Article Title: Dual membrane receptor degradation via folate receptor targeting chimera

doi: 10.1038/s41467-025-63882-5

Figure Lengend Snippet: a Schematic of pharmacokinetic studies in mice following intraperitoneal (i.p.) injection of EGFR/HER2 FolTAC-dual v1.0. b Western blot analysis of compound levels in mice plasma at defined time points. Band intensities were quantified, and data are presented as mean ± SD with biological repeats ( n = 3). c Diagram of PDO workflow for functional testing. d Representative images of EdU (red) and DAPI (blue) staining in PDOs from patients 8 and 10 after 7-day treatment, with the drug refreshed every two days. Magenta marks EdU-positive nuclei. CTRL: control. Scale bar, 40 μm. e – f Quantification of EdU-positive cells in PDOs from patient 8 ( e ) and patient 10 ( f ), treated with vehicle (CTRL), 25, 100, or 250 nM EGFR/HER2 FolTAC-dual v1.0. Each dot represents an individual PDO structure quantified in one biological replicate, and the violin plots represent data distribution from a representative independent experiment out of three repeats. Sample sizes were: CTRL ( n = 27), 25 nM ( n = 25), 100 nM ( n = 27), and 250 nM ( n = 27) for patient 8; CTRL ( n = 28), 25 nM ( n = 27), 100 nM ( n = 33), and 250 nM ( n = 31) for patient 10. Data are shown as mean ± SD. Statistical significance was assessed using two-sided unpaired Student’s t-tests. Exact p -values are indicated above the plots. *, **, **** indicate p < 0.05, p < 0.01, p < 0.0001, respectively; ns, not significant. g Western blot analysis showing degradation of EGFR and HER2 in PDOs from patient 8 following treatment. h Experimental scheme for in vivo efficacy testing. i Representative tumor images from control (PBS, n = 7), and treated (Trastuzumab, n = 7; HER2 FolTAC, n = 7; HER2 FolTAC+EGFR FolTAC, n = 7; EGFR/HER2 FolTAC-dual v1.0, n = 7) groups at endpoint. j Tumor growth in Traz/Lap-resistant SKBR3 xenografts following i.p. injection (5 mg/kg). Two-sided unpaired Student’s t-tests were used. Exact p-values are shown above the bars. p < 0.05 considered significant; ** p < 0.01; ns, not significant. Created in BioRender. WANG, Z. (2025) https://BioRender.com/d5n9vj6 .

Article Snippet: Binding affinity measurements of HER2 affibody-Fc and Trastuzumab in both SKBR3-WT and Traz/Lap-resistant SKBR3 cells revealed no significant difference (Supplementary Fig. ), suggesting that the enhanced efficacy of FolTAC-dual may arise from its cooperative configuration or multivalent engagement, rather than from intrinsic differences in binding strength.

Techniques: Injection, Western Blot, Clinical Proteomics, Functional Assay, Staining, Control, In Vivo