lag3  (Proteintech)

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Design of a SHEDTAC library spanning five anti-ADAM10 VHH and three anti-LAG3 VHH, all targeting distinct epitopes. Diverse configurations are achieved through N→C or C→N terminal VHH fusions, affording a library of thirty unique LAG3/ADAM10 bispecific combinations. ( b ) Reducing SDS-PAGE analysis of purified SHEDTACs used to treat cells in ( c,d ). ( c ) T cell surface LAG3 shedding by the protease ADAM10, which is accelerated by SHEDTACs ( d ) Western blot analysis of T cell pellets indicating levels of intact LAG3 on cells following 24h treatment, quantified in ( e ). Intensity is expressed as percent of vehicle (V). “+” indicates the addition of ionomycin (10µg/ml) to induce ADAM10 activity.

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: SDS Page, Purification, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Reducing SDS-PAGE indicating SHEDTAC#8, selected for its high activity shown in . ( b-e ) Flow cytometry contour plots showing LAG3 abundance on CD3+ADAM10+ PBMCs following 1h treatment with ( b ) vehicle, ( c ) 500nM SHEDTAC, ( d,e ) equimolar TEV-proteolyzed SHEDTAC, serving as monospecific controls. ( f ) Flow cytometry contour plots showing LAG3 abundance on CD3+ADAM10+ PBMCs treated with SHEDTAC (left) or equimolar TEV-digested SHEDTAC (right). Prior to treatment, cells were incubated for 2h at 37°C with vehicle (left plots), proteasome inhibitor (MG132, 10µM, middle plots), or lysosome inhibitor (Dynasore, 50µM, right plots). ( g ) Western blot analysis of cell pellets and conditioned cell supernatants treated with SHEDTAC, sampled every 10 minutes for 60 minutes, indicating time-dependent decreases in full-length (∼70kDa) LAG3 and concomitant increases in soluble LAG3 (sLAG3, ∼60kDa) ectodomain released into the growth medium by ADAM10. ( h ) quantification of data from ( g ) normalized to GAPDH and expressed as percent control of cell pellet at t=0. ( i ) Cells from ( g ) following 24h SHEDTAC treatment. ( j ) Ratio of LAG3:ADAM10 as determined by flow cytometry over a range of SHEDTAC concentrations.

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: SDS Page, Activity Assay, Flow Cytometry, Incubation, Western Blot, Control

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) LAG3 suppresses T cell signaling through homodimer formation, and interactions with the TCR on T cells and MHCII on antigen presenting cells (APCs) (left). LAG3-SHEDTACs catalyze LAG3 proteolysis by endogenous protease ADAM10 to restore TCR signaling and induce a luciferase reporter (right). ( b ) Flow cytometry contour plots indicating LAG3 abundance on ADAM10(+) luciferase reporter Jurkat cells treated with isotype control or LAG3-SHEDTAC. ( c ) Dose-dependent luminescence increases following treatment with SHEDTAC at the indicated concentration, illustrating enhanced TCR signaling that is afforded through LAG3 shedding. RLU = relative luminescence units

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Luciferase, Flow Cytometry, Control, Concentration Assay

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Gating strategy for activated CD3+ADAM10+LAG3+ PBMCs

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques:

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Flow cytometry TEV-normalization scheme. SHEDTACs were normalized to their equimolar TEV-proteolyzed controls, and significant shedding was indicated wherever this ratio ‘x’ was x<1. In contrast, TEV-normalized shedding where x≥1 indicates low SHEDTAC activity, or VHH competition with LAG3 detection reagents, confirmed by western blot

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Flow Cytometry, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Comparison of western blot versus flow cytometry analyses to assess LAG3 shedding by ADAM10 following treatment with SHEDTACs

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Comparison, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Soluble LAG3 (sLAG3) generation through receptor shedding. ( b ) primary amino acid sequence analysis showing transmembrane and intracellular regions totaling ∼8.8kDa. ( c ) Dose-dependent loss of LAG3 abundance on T cells following treatment with SHEDTACs. Contour plots correspond to data plotted in . ( d ) Concomitant soluble LAG3 production in conditioned supernatants from cells treated in ( c ).

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Sequencing

Journal: Materials Today Bio

Article Title: Activated T-cell membrane-derived nanocargoes displaying multi-immune checkpoints for enhanced cancer immunotherapy

doi: 10.1016/j.mtbio.2025.102702

Figure Lengend Snippet: Association between intratumoural STING signature and ICs expression. (A–H) Representative IHC image and quantification of the expression level of STING and ICs including PD-L1/PD-1, GAL-9/TIM-3 and GAL-3/LAG-3 in lung cancer patients. (I) Experimental timeline and indicated treatment groups (three intravenous injections, diABZI: 0.75 mg per kg (body weight), n = 6 mice). (J) Statistical analysis tumor volume on day 13 of mice with CT-26 tumors receiving the indicated treatments. (K–O) Representative flow cytometric densities of total immune cells (CD45 + ), T lymphocytes (CD3 + ), DCs (CD11c + MHC-II + ), Macrophages (CD11b + F4/80 + ) and NK cells (CD3 − CD49b + ) in the tumors receiving diABZI or PBS at three days after the last injections (n = 6 mice). (P–R) Representative flow cytometric quantification of the expression level of PD-1, TIM-3 and LAG-3 in tumor-infiltrating CD3 + T cells of CT-26-bearing mice receiving diABZI or PBS at three days after the last injections (n = 6 mice). All data are presented as the mean ± S.D. The p values were determined by two-tailed Student's t -test for (B–H) and (J–R).

Article Snippet: 66220-1-Ig), LAG-3 Polyclonal antibody (Proteintech:16616-1-AP), and Recombinant Anti-TIM-3 antibody (Abcam: EPR22241 ), PD-L1 Rabbit Polyclonal Antibody (Abiowell: cat.no.

Techniques: Expressing, Two Tailed Test

Journal: Materials Today Bio

Article Title: Activated T-cell membrane-derived nanocargoes displaying multi-immune checkpoints for enhanced cancer immunotherapy

doi: 10.1016/j.mtbio.2025.102702

Figure Lengend Snippet: Screening activated T-cell with high expression of ICs. (A) Diagram of activated T cells with multiple ICs screening. (B–D) Representative flow cytometric histograms (left) and curves (right) of the expression level of ICs including PD-1 (B), LAG-3 (C) and TIM-3 (D) of activated T cells from day 0–8 of the T cell screening assay, gated on CD3 + T cells, CD4 + T cells, CD8 + T cells (n = 3 independent experiments), MFI, mean fluorescence intensity. (E) The KEGG pathway enrichment analysis of all types of membrane-associated genes that are upregulated in activated T cells compared with un-activated T cells. (F) Heatmap showing the scaled expression of membrane-associated genes in activated T cells compared with un-activated T cells. All data are presented as the mean ± s.d.

Article Snippet: 66220-1-Ig), LAG-3 Polyclonal antibody (Proteintech:16616-1-AP), and Recombinant Anti-TIM-3 antibody (Abcam: EPR22241 ), PD-L1 Rabbit Polyclonal Antibody (Abiowell: cat.no.

Techniques: Expressing, Screening Assay, Fluorescence, Membrane

Journal: Materials Today Bio

Article Title: Activated T-cell membrane-derived nanocargoes displaying multi-immune checkpoints for enhanced cancer immunotherapy

doi: 10.1016/j.mtbio.2025.102702

Figure Lengend Snippet: The preparation and characterizations of AM-dLNPs. (A)Transmission electron microscopy images of non-activated T cells membrane vesicles (M) and activated T cells membrane vesicles (AM). Scale bars, 100 nm. (B) Western blot analysis of CD3 protein in activated T cell (AT) and AM. (C, D) The average hydrodynamic diameter and surface zeta potential of AM and M as measured by dynamic light scattering (n = 3 independent experiments). (E) Rescue of ICs-mediated T-cell exhaustion by AM in cocultures of IFN-γ-pretreated (20 ng/ml, 24 h) 4T1-OVA/Luci cells and OT-1 T cells at an effector: target ratio of 5:1. 4T1-OVA/Luci cells death in the cocultures was evaluated by luciferase reporter assay system (n = 3 independent experiments). (F) Representative flow cytometric percentage of IFN-γ+, Perforin + and Granzyme B + (CzmB) in OT-1 T cells after co-incubation with IFN-γ-pretreated 4T1-OVA/Luci cells and diABZI, dLNPs, M-dLNPs, AM-LNPs, AM-dLNPs or PBS for 24h at an effector: target ratio of 5:1. (G–J) The average hydrodynamic diameter (G, H), polydispersity (I) and surface zeta potential (J) of T-cell membrane (TM), dLNPs, M-dLNPs, AM-LNPs and AM-dLNPs, as measured by dynamic light scattering (n = 3 independent experiments). (K) Western blot analysis of CD3, PD-1, LAG-3, and TIM-3 in dLNPs, M-dLNPs, AM-LNPs and AM-dLNPs. (L) Expression of PD-L1 on AM-derived nanocargoes was determined by Flow NanoAnalyzer. (M) Expression of TIM-3 on AM-derived nanocargoes was determined by Flow NanoAnalyzer. (N) Transmission electron microscopy images of dLNPs, M-dLNPs, AM-LNPs and AM-dLNPs. Scale bars, 200 nm. (O) High-performance liquid chromatography analysis of diABZI concentrations in dLNPs, M-dLNPs, AM-LNPs and AM-dLNPs (n = 3 independent experiments). dLNPs: diABZI-loaded liposomes, M-dLNPs: non-actived T-cell membrane-decorated diABZI-loaded liposomes, AM-LNPs: actived T-cell membrane-decorated empty liposomes, AM-dLNPs: actived T-cell membrane-decorated diABZI-loaded liposomes. All the data are presented as the mean ± S.D. The p values were determined by one-way ANOVA with Tukey's post-test for (E).

Article Snippet: 66220-1-Ig), LAG-3 Polyclonal antibody (Proteintech:16616-1-AP), and Recombinant Anti-TIM-3 antibody (Abcam: EPR22241 ), PD-L1 Rabbit Polyclonal Antibody (Abiowell: cat.no.

Techniques: Transmission Assay, Electron Microscopy, Membrane, Western Blot, Zeta Potential Analyzer, Luciferase, Reporter Assay, Incubation, Expressing, Derivative Assay, High Performance Liquid Chromatography, Liposomes

Journal: Materials Today Bio

Article Title: Activated T-cell membrane-derived nanocargoes displaying multi-immune checkpoints for enhanced cancer immunotherapy

doi: 10.1016/j.mtbio.2025.102702

Figure Lengend Snippet: AM-dLNPs induced potent anti-tumor immune responses. (A–B) Representative flow cytometry plots (left) and statistical analysis (right) of T cell exhaustion markers PD-1 (A) and LAG-3 (B) on CD3 + T cells across different treatment groups by flow cytometry (n = 5 mice). (C–D) Representative flow cytometric plots and frequencies of Perforin (C) and Gzm B (D) that generated by tumor-infiltrating CD8 + T cells harvested on day 3 from bearing CT-26 mice after the last injection of the three-injection regimen. (n = 5 mice). (E) Representative IF images of tumor sections obtained from mice receiving three injections of the indicated treatments. Gzm B (green) and Perforin (red) were visualized using fluorescence-labelled antibodies. The nuclei were stained with DAPI (blue). Scale bars, 25 μm. (F) Representative flow cytometry plots and statistical analysis of naïve T cells (CD44 − CD62L + ), central memory T cells (CD44 + CD62L + , TCM), effector memory T cells (CD44 + CD62L − , TEM) and terminal exhausted T cells (TE X ) within the TME across different treatment groups (n = 5 mice). (G, H) Representative flow cytometric frequencies of CD86 + DCs (G) and NK cells (H) in spleen harvested on day 3 from CT-26-bearing mice after the last injection of the three-injection regimen (n = 5 mice). All the data are expressed as mean ± S.D. The p values were determined by one-way ANOVA with Tukey's post-test for (A–D), (F) and (G, H). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 66220-1-Ig), LAG-3 Polyclonal antibody (Proteintech:16616-1-AP), and Recombinant Anti-TIM-3 antibody (Abcam: EPR22241 ), PD-L1 Rabbit Polyclonal Antibody (Abiowell: cat.no.

Techniques: Flow Cytometry, Generated, Injection, Fluorescence, Staining