Journal: Biomaterials Research

Article Title: PD-L1/Lag3 Bispecific Immune Checkpoint Blocking Nanocage Exhibits Potent Antitumor Activity beyond Dual Blockade of PD-L1 and Lag3

doi: 10.34133/bmr.0362

Figure Lengend Snippet: (A) Three-dimensional (3D) models of the 3 types of peptide-displaying ferritin nanocages. (B) Schematic representation of Lag3pep-ferritin nanocages. Lag3pep1 (CIRNDPAVC) or Lag3pep2 (CSVLNASGC) was fused to the N-terminus (N1 and N2), the C-terminus (C1 and C2), or the loop region of ferritin (L1 and L2). (C) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified Lag3pep-ferritin nanocages. (D) Human embryonic kidney (HEK) 293T cells expressing lymphocyte-activation gene 3 (Lag3) were incubated with Lag3pep-ferritin nanocages or wild-type ferritin heavy chain (wFTH) at 4 °C for 1 h. Binding was detected using an anti-ferritin antibody (red), nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue), and green fluorescent protein (GFP)-tagged Lag3 expression is shown in green. Scale bars: 30 μm. (E) Surface plasmon resonance (SPR) analysis showing the binding affinity of Lag3pep-ferritin nanocages to Lag3. Resonance units (RU) at 500 nM of each construct are shown, depicting association and dissociation kinetics.

Article Snippet: THP-1 cells expressing human leukocyte antigen-DR isotype (HLA-DR, a subtype of human MHC-II) were used to assess whether Lag3-targeting ferritin nanocages could block interaction between Lag3 and HLA-DR. THP-1 cells were stimulated with 50 ng/ml interferon-gamma (IFN-γ) (PeproTech) for 48 h and then incubated with 400 ng of hLag3-Fc protein (18.3 nM; Acro Biosystems) in the presence or absence of Lag3pep-ferritin nanocages (L1, L2, P1L1, or P1L2; 183 nM) at 4 °C for 30 min. As a positive control, the same molar concentration (183 nM) of anti-human Lag3 blocking antibody (AdipoGen Life Sciences) was used.

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Expressing, Activation Assay, Incubation, Binding Assay, SPR Assay, Construct

Journal: Biomaterials Research

Article Title: PD-L1/Lag3 Bispecific Immune Checkpoint Blocking Nanocage Exhibits Potent Antitumor Activity beyond Dual Blockade of PD-L1 and Lag3

doi: 10.34133/bmr.0362

Figure Lengend Snippet: Construction and characterization of programmed cell death ligand 1 (PD-L1)/Lag3 bispecific ferritin nanocages. (A) Schematic representation of PD-L1/Lag3 bispecific ferritin nanocages (P1L1 and P1L2) and their parental nanocages (P1, L1, and L2). (B) SDS-PAGE analysis of purified PD-L1/Lag3 bispecific ferritin nanocages. (C) Three-dimensional model of the PD-L1/Lag3 bispecific ferritin nanocage generated by computational simulation. (D) Dynamic light scattering (DLS) analysis showing the size distribution of PD-L1/Lag3 bispecific ferritin nanocages. (E) Transmission electron microscopy (TEM) images confirming the cage structure and uniform size of the PD-L1/Lag3 bispecific ferritin nanocages.

Article Snippet: THP-1 cells expressing human leukocyte antigen-DR isotype (HLA-DR, a subtype of human MHC-II) were used to assess whether Lag3-targeting ferritin nanocages could block interaction between Lag3 and HLA-DR. THP-1 cells were stimulated with 50 ng/ml interferon-gamma (IFN-γ) (PeproTech) for 48 h and then incubated with 400 ng of hLag3-Fc protein (18.3 nM; Acro Biosystems) in the presence or absence of Lag3pep-ferritin nanocages (L1, L2, P1L1, or P1L2; 183 nM) at 4 °C for 30 min. As a positive control, the same molar concentration (183 nM) of anti-human Lag3 blocking antibody (AdipoGen Life Sciences) was used.

Techniques: SDS Page, Purification, Generated, Transmission Assay, Electron Microscopy

Journal: Biomaterials Research

Article Title: PD-L1/Lag3 Bispecific Immune Checkpoint Blocking Nanocage Exhibits Potent Antitumor Activity beyond Dual Blockade of PD-L1 and Lag3

doi: 10.34133/bmr.0362

Figure Lengend Snippet: In vitro binding of PD-L1/Lag3 bispecific ferritin nanocages. (A) MDA-MB-231 cells were incubated with P1, P1L1, P1L2, or wFTH at 4 °C for 1 h. Binding interactions were detected using an anti-ferritin antibody (green), and nuclei were counterstained with DAPI (blue). Scale bars: 30 μm. (B) HEK 293T cells expressing Lag3 were incubated with P1L1, P1L2, L1, L2, or wFTH at 4 °C for 1 h. Binding was visualized using an anti-ferritin antibody (red), GFP-Lag3 expression is shown in green, and nuclei were counterstained with DAPI (blue). Scale bars: 30 μm. (C) Jurkat T cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin, and chloroquine to express Lag3 followed by incubation with P1L1, P1L2, L1, L2, or wFTH. Bound proteins were measured by anti-ferritin antibody with flow cytometric analysis. Statistical comparisons were conducted with Lag3pep displaying nanocages against wFTH (*** P < 0.001; one-way analysis of variance [ANOVA]); nonsignificant differences are not shown. (D) SPR analysis of Lag3pep-displaying ferritin constructs (L1, L2, P1L1, and P1L2) against Lag3-coated surface. RU were measured at varying protein concentrations to determine binding affinities ( K D ). (E) SPR analysis of P1L2 against PD-L1-coated surface. RU were measured at varying protein concentrations to determine binding affinities ( K D ).

Article Snippet: THP-1 cells expressing human leukocyte antigen-DR isotype (HLA-DR, a subtype of human MHC-II) were used to assess whether Lag3-targeting ferritin nanocages could block interaction between Lag3 and HLA-DR. THP-1 cells were stimulated with 50 ng/ml interferon-gamma (IFN-γ) (PeproTech) for 48 h and then incubated with 400 ng of hLag3-Fc protein (18.3 nM; Acro Biosystems) in the presence or absence of Lag3pep-ferritin nanocages (L1, L2, P1L1, or P1L2; 183 nM) at 4 °C for 30 min. As a positive control, the same molar concentration (183 nM) of anti-human Lag3 blocking antibody (AdipoGen Life Sciences) was used.

Techniques: In Vitro, Binding Assay, Incubation, Expressing, Construct

Journal: Biomaterials Research

Article Title: PD-L1/Lag3 Bispecific Immune Checkpoint Blocking Nanocage Exhibits Potent Antitumor Activity beyond Dual Blockade of PD-L1 and Lag3

doi: 10.34133/bmr.0362

Figure Lengend Snippet: Cellular binding and blocking activity of Lag3pep-displaying and PD-L1/Lag3 bispecific ferritin nanocages. (A) Schematic of the cell-based blocking assay using HLA-DR-expressing THP-1 cells to evaluate the ability of Lag3pep-displaying ferritin nanocages to inhibit the interaction between Lag3 protein and its ligand HLA-DR. (B) Flow cytometry quantification of human recombinant Lag3-Fc binding to HLA-DR-expressing THP-1 cells in the presence of either an anti-human Lag3 blocking antibody (a-hLag3) or Lag3pep-displaying ferritin nanocages. Mean fluorescence intensities are shown. Data are presented as mean ± SD (*** P < 0.001; one-way ANOVA); nonsignificant differences are not shown. (C) Mouse colon cancer cells (MC38) and mouse glioma cells (CT-2A and GL26) were incubated with P1L2 or wFTH at 4 °C for 1 h. Binding was detected using an anti-ferritin antibody (green), and nuclei were counterstained with DAPI (blue). Scale bars: 30 μm.

Article Snippet: THP-1 cells expressing human leukocyte antigen-DR isotype (HLA-DR, a subtype of human MHC-II) were used to assess whether Lag3-targeting ferritin nanocages could block interaction between Lag3 and HLA-DR. THP-1 cells were stimulated with 50 ng/ml interferon-gamma (IFN-γ) (PeproTech) for 48 h and then incubated with 400 ng of hLag3-Fc protein (18.3 nM; Acro Biosystems) in the presence or absence of Lag3pep-ferritin nanocages (L1, L2, P1L1, or P1L2; 183 nM) at 4 °C for 30 min. As a positive control, the same molar concentration (183 nM) of anti-human Lag3 blocking antibody (AdipoGen Life Sciences) was used.

Techniques: Binding Assay, Blocking Assay, Activity Assay, Expressing, Flow Cytometry, Recombinant, Fluorescence, Incubation

Journal: Biomaterials Research

Article Title: PD-L1/Lag3 Bispecific Immune Checkpoint Blocking Nanocage Exhibits Potent Antitumor Activity beyond Dual Blockade of PD-L1 and Lag3

doi: 10.34133/bmr.0362

Figure Lengend Snippet: (A) Schematic of the experiment to evaluate the efficacy of P1L2 in enhancing CD8 + T-cell activity. CD8 + T cells were isolated from the spleens of MC38 tumor-bearing mice, activated, and cocultured with MC38 tumor cells at a T:MC38 ratio of 10:1 for 24 h. Treatments included anti-mouse PD-L1 or Lag3 antibodies (10 μg/ml), ferritin constructs (50 nM), or no treatment. (B) T-cell proliferation was assessed via carboxyfluorescein succinimidyl ester (CFSE) dilution after 24 h of coculture. (C and D) Interferon-gamma (IFN-γ) (C) and Granzyme B (GZMB) (D) levels in the supernatant were quantified by enzyme-linked immunosorbent assay (ELISA). (E) Lactate dehydrogenase (LDH) release was measured as an indicator of tumor cell death. Bar graphs represent mean ± SD. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns; not significant.

Article Snippet: THP-1 cells expressing human leukocyte antigen-DR isotype (HLA-DR, a subtype of human MHC-II) were used to assess whether Lag3-targeting ferritin nanocages could block interaction between Lag3 and HLA-DR. THP-1 cells were stimulated with 50 ng/ml interferon-gamma (IFN-γ) (PeproTech) for 48 h and then incubated with 400 ng of hLag3-Fc protein (18.3 nM; Acro Biosystems) in the presence or absence of Lag3pep-ferritin nanocages (L1, L2, P1L1, or P1L2; 183 nM) at 4 °C for 30 min. As a positive control, the same molar concentration (183 nM) of anti-human Lag3 blocking antibody (AdipoGen Life Sciences) was used.

Techniques: Activity Assay, Isolation, Construct, Enzyme-linked Immunosorbent Assay

Journal: Biomaterials Research

Article Title: PD-L1/Lag3 Bispecific Immune Checkpoint Blocking Nanocage Exhibits Potent Antitumor Activity beyond Dual Blockade of PD-L1 and Lag3

doi: 10.34133/bmr.0362

Figure Lengend Snippet: (A) Experimental design for antitumor treatment. MC38 syngeneic colon tumor cells were subcutaneously implanted into mice, and treatment began once tumor volumes reached approximately 50 to 100 mm 3 . P1L2, P1, L2, P1 + L2, or wFTH were administered intravenously 3 times weekly, while anti-PD-L1 or anti-Lag3 antibodies were injected intraperitoneally twice weekly. (B and C) Tumor growth curves during treatment. Statistical significance was determined using 2-way ANOVA followed by Dunnett’s multiple comparison test (* P < 0.05, ** P < 0.01, *** P < 0.001); nonsignificant differences are not shown. (D) Final tumor volumes at the end of the study, showing significant inhibition with P1L2 ( **P < 0.01). Data are presented as mean ± SE (* P < 0.05, ** P < 0.01; t test). (E) Body weight changes (ns, not significant; 2-way ANOVA followed by Dunnett’s multiple comparison test). (F) Flow cytometry analysis of CD8 + , Treg (FoxP 3+ ), and ratio of CD8 + /Treg cells in tumor tissues ( n = 5 per group). Data are shown as mean ± SE (* P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant; one-way ANOVA).

Article Snippet: THP-1 cells expressing human leukocyte antigen-DR isotype (HLA-DR, a subtype of human MHC-II) were used to assess whether Lag3-targeting ferritin nanocages could block interaction between Lag3 and HLA-DR. THP-1 cells were stimulated with 50 ng/ml interferon-gamma (IFN-γ) (PeproTech) for 48 h and then incubated with 400 ng of hLag3-Fc protein (18.3 nM; Acro Biosystems) in the presence or absence of Lag3pep-ferritin nanocages (L1, L2, P1L1, or P1L2; 183 nM) at 4 °C for 30 min. As a positive control, the same molar concentration (183 nM) of anti-human Lag3 blocking antibody (AdipoGen Life Sciences) was used.

Techniques: Injection, Comparison, Inhibition, Flow Cytometry

Journal: Parasites & Vectors

Article Title: LAG3 constrains anti-parasitic response by effector CD4 + T-cell in early Echinococcus multilocularis -infected mice

doi: 10.1186/s13071-026-07246-y

Figure Lengend Snippet: LAG3 blockade promotes early clearance of E. multilocularis in mouse liver. A Establishment of a LAG3-neutralizing antibody model in vivo. B Representative hematoxylin–eosin staining image (left panel ×40, enlarged ×100 on the right panel) in the liver tissue sections of E. multilocularis -infected mice treated with IgG and anti-LAG3 mAb for 4 weeks. C Percentage and area of different lesion types in the liver of E. multilocularis -infected mice treated with IgG ( n = 7) and anti-LAG3 mAb ( n = 5) for 4 weeks. D Representative Sirius red staining image (left panel ×100, enlarged ×200 on the right panel) in the liver tissue sections of E. multilocularis -infected mice treated with IgG and anti-LAG3 mAb for 4 weeks. E Percentage of different lesion types in the liver of E. multilocularis -infected mice treated with IgG ( n = 7) and anti-LAG3 mAb ( n = 5) for 4 weeks. F Percentage of CD4 + T cells, CD4 + Tn, and CD4 + Teff in the liver and spleen of E. multilocularis -infected mice treated with IgG ( n = 5) and anti-LAG3 mAb ( n = 6) for 4 weeks. G Absolute number of CD4 + T cells, CD4 + Tn, and CD4 + Teff in the liver and spleen of E. multilocularis -infected mice treated with IgG ( n = 5) and anti-LAG3 mAb ( n = 6) for 4 weeks. All data are presented as mean + SD. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., P > 0.05

Article Snippet: Wild-type C57BL/6 mice were administered 200 μg of anti-LAG3 monoclonal antibody (mAb) (clone C9B7W, BioXCell) or an isotype control (Rat IgG1, κ, BioXCell) 2 days and 1 day prior to E. multilocularis infection via intraperitoneal injection (i.p.), followed by subsequent injections of 200 μg antibody or isotype control every 3 days thereafter.

Techniques: In Vivo, Staining, Infection

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: bioRxiv

Article Title: Immune checkpoint inhibitors amplify type 2 immune mediated repair by pro-regenerative scaffolds

doi: 10.64898/2026.01.31.703034

Figure Lengend Snippet: (A) Frequency of IL4 + T H 2 out of CD4 + T cells with isotype, monotherapy anti-PD1, monotherapy anti-CTLA4, or combination anti-PD1/anti-CTLA4 in ECM-treated injuries. (B) Frequency of IL4 + T H 2 out of CD4 + T cells with no treatment, isotype, or monotherapy anti-LAG3 (standard dose, 5 mg/kg) in saline-and ECM-treated injuries. (C) Frequency of IL4 + T H 2 out of CD4 + T cells with isotype or monotherapy anti-LAG3 (increased dose, 10 mg/kg) in saline-and ECM-treated injuries. Data presented as mean±SD and analyzed using one-way ANOVA with Dunnett’s multiple comparison relative to isotype control (A) or two-way ANOVA with Tukey’s multiple comparisons test (B-C). NS p>0.05; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Endogenous peroxidase was blocked then anti-LAG3 clone 17B4 (LS-C344932-100, LSBio, Shirley, MA, RRID: AB_3076336) was applied for 60 min at a concentration of 0.025 μg/mL at room temperature.

Techniques: Saline, Comparison, Control