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