recombinant e6 protein  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Engineered human B cells targeting tumor-associated antigens exhibit antigen presentation and antibody-mediated functions

doi: 10.3389/fimmu.2025.1621222

Figure Lengend Snippet: Engineered B cells activate BCR signaling cascades and secrete isotype-switched antigen-specific antibodies. (A) Representative immunoblot of B cells engineered to express either C1P5 or 6F4 E6-binding BCRs, stimulated with recombinant E6 protein, anti-IgM/IgG protein control or unstimulated. Protein extracts were analyzed using anti-pERK antibody (top panel) or anti-total ERK antibody (bottom panel). (B) Quantification of mean ratio expression of ERK to pERK as in (A) ns=pv>0.05, *=pv<0.05, **=pv<0.01 for two-way ANOVA with Dunnett’s multiple comparisons test. (C) Same as A for the CLDN6-binding BCRs AB37 or IMAB206. Cell stimulation occurred with recombinant CLDN6 protein. (D) Quantification of experiments as in (C) For (A-D) , data representative of 2–3 experiments with 2 donor cells. ns=pv>0.05, **=pv<0.01 for two-way ANOVA with Dunnett’s multiple comparisons test. (E) Quantification of IgG antibodies in the supernatants of engineered B cells expressing either C1P5, AB37 or IMAB206, by ELISA. ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (F) Representative ELISPOT images of E6-specific IgM, IgG and IgA secreted from B cells engineered to express the anti-E6 6F4 antibody. (G) Quantification of data as in (F) as normalized to the total amount of cells seeded in the wells. ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. For (E-G) pooled data from 2–3 experiments with 2–4 donor cells. For (B, D, E) , (G) mean and error bars are indicated, each dot represents an independent experiment.

Article Snippet: Recombinant E6 protein was from R&D Systems.

Techniques: Western Blot, Binding Assay, Recombinant, Control, Expressing, Cell Stimulation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: Engineered human B cells targeting tumor-associated antigens exhibit antigen presentation and antibody-mediated functions

doi: 10.3389/fimmu.2025.1621222

Figure Lengend Snippet: Engineered B cells and immune complexes taken up by dendritic cells present E6 antigen to activate E6-specific MHC class II-restricted T cells. (A) Schema depicting the engineered B cell antigen presentation assay. B cells, either non-engineered (EP-only) or engineered to express anti-E6 antibodies (6F4 or C1P5), are loaded with recombinant E6 antigen at multiple concentrations and then incubated with T cells, either engineered (Class II TCR) or non-engineered (UTD) to express an anti-E6 TCR. The concentration of IFNγ in the supernatants is then analyzed by ELISA. (B) ELISA for IFNγ as in (A) Data representative of 2 experiments with 2 donors. Bars and error bars represent mean and SD. ns=pv>0.05, ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (C) Schema depicting the immune complex-mediated antigen presentation assay. Immune complexes were formed with concentrated antibodies from the supernatants of B cells, either non-engineered or engineered to express anti-E6 antibodies, and recombinant E6 antigen at multiple concentrations. These were then incubated with myeloid-derived dendritic cells and then co-cultured with T cells, either engineered or non-engineered to express an anti-E6 TCR. The concentration of IFNγ in the supernatants is then analyzed by ELISA. (D) ELISA for IFNγ as in (C) Data representative of 2 experiments with 2 donors. Bars and error bars represent mean and SD. ns=pv>0.05, *=pv<0.05, ***=pv<0.001, ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (E) Schema depicting the flow cytometry-based engineered B cell membrane-bound antigen uptake assay. Engineered or non-engineered B cells are incubated with labeled tumor cells either expressing or not expressing the targeted antigen. The frequency of B cells with the tumor label is then analyzed by flow cytometry. (F) Flow cytometry results as in (E) for B cells engineered to express anti-CLDN6 IMAB206 (blue) or anti-E6 (6F4) antibodies (grey). ns=pv>0.05, ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (G) Flow cytometry results as in (E) for B cells engineered to express anti-E6 6F4 antibodies (orange), or anti-CLDN6 IMAB206 antibodies (grey). ns=pv>0.05, **=pv<0.01, for two-way ANOVA with Tukey’s multiple comparisons test. (H) Schema depicting a flow cytometry-based engineered B cell antigen presentation assay. B cells, either non-engineered or engineered to express anti-E6 antibodies, were incubated with tumor cells, engineered to express the membrane-bound E6 antigen, and with T cells, either engineered or non-engineered to express an anti-E6 TCR. The frequency of T cells expressing intracellular IFNγ was then analyzed by flow cytometry. (I) Flow cytometry results as in (H) for B cells engineered to express anti-E6 6F4 (orange) antibodies. Data representative of 3 experiments using one donor. Bars and error bars represent mean and SD. ns=pv>0.05, ****=pv<0.0001, for two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Recombinant E6 protein was from R&D Systems.

Techniques: Immunopeptidomics, Recombinant, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Flow Cytometry, Membrane, Labeling, Expressing

Journal: Frontiers in Immunology

Article Title: Engineered human B cells targeting tumor-associated antigens exhibit antigen presentation and antibody-mediated functions

doi: 10.3389/fimmu.2025.1621222

Figure Lengend Snippet: Engineered B cells activate BCR signaling cascades and secrete isotype-switched antigen-specific antibodies. (A) Representative immunoblot of B cells engineered to express either C1P5 or 6F4 E6-binding BCRs, stimulated with recombinant E6 protein, anti-IgM/IgG protein control or unstimulated. Protein extracts were analyzed using anti-pERK antibody (top panel) or anti-total ERK antibody (bottom panel). (B) Quantification of mean ratio expression of ERK to pERK as in (A) ns=pv>0.05, *=pv<0.05, **=pv<0.01 for two-way ANOVA with Dunnett’s multiple comparisons test. (C) Same as A for the CLDN6-binding BCRs AB37 or IMAB206. Cell stimulation occurred with recombinant CLDN6 protein. (D) Quantification of experiments as in (C) For (A-D) , data representative of 2–3 experiments with 2 donor cells. ns=pv>0.05, **=pv<0.01 for two-way ANOVA with Dunnett’s multiple comparisons test. (E) Quantification of IgG antibodies in the supernatants of engineered B cells expressing either C1P5, AB37 or IMAB206, by ELISA. ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (F) Representative ELISPOT images of E6-specific IgM, IgG and IgA secreted from B cells engineered to express the anti-E6 6F4 antibody. (G) Quantification of data as in (F) as normalized to the total amount of cells seeded in the wells. ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. For (E-G) pooled data from 2–3 experiments with 2–4 donor cells. For (B, D, E) , (G) mean and error bars are indicated, each dot represents an independent experiment.

Article Snippet: For soluble controls, 1e5 B cells were added to a 96-well plate and incubated with 0 or 100 nM E6 protein (R&D Systems) or 1 μg/well E6 1–15 peptide (Genscript) for 20 minutes, followed by the addition of 1e5 UTD or E6 TCR T cells for a 1:1 ratio of B Cells:T cells.

Techniques: Western Blot, Binding Assay, Recombinant, Control, Expressing, Cell Stimulation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: Mouse B cells engineered to express an anti-HPV antibody elicit anti-tumor T cell responses

doi: 10.3389/fimmu.2025.1613879

Figure Lengend Snippet: Immune complexes derived from antibodies secreted by EBCs activate T cells. (A) Isotype-specific ELISA for antibodies against E6 in the supernatants of B cells engineered as in Figure 2A . Mean OD values and SD are shown for IgG (left) or IgM (right) E6-specific antibodies from EBC (orange) or EPOnly (black) cells, n = 2–3. (B) Quantification of IgG data from (A) using a recombinant IgG1 antibody. Mean and SD values are represented. Pooled data from two independent experiments, n = 2. (C) Representative PCR on reverse-transcribed mRNA from EBCs. Primer binding sites are indicated with arrows. The anti-E6 engineering cassette are shown in orange, and endogenous segments in white or gray. For each amplicon, the DNA (top) or mRNA (bottom) are illustrated. Only spliced fragments derived from mRNA are amplified due to polymerization length constraints. (D) Gel electrophoresis of amplicons from (C) , using RNA extracted from EBCS (left) or mRNA from EPOnly control B cells (right). Molecular weight ladders with relative sizes are shown beside each gel. (E) Representative schematic of cell sources used in (F–I) . Bone marrow from naïve mice was used to generate myeloid-derived dendritic cells. T cells were obtained from mice immunized with E6. B cells for engineering were isolated from separate naïve mice. (F) Representative experimental scheme for (G–I) . Myeloid-derived dendritic cells were seeded and loaded with immune complexes formed by incubating E6 antigen with EBC-derived supernatant. T cells from E6-immunized mice were then added to the culture. (G) Intracellular flow cytometry for IFNg in CD8 + T cells from cultures as in (F) , incubated with supernatants from either EBCs (orange) or EPOnly control B cells (black). Pre-gated on singlets, live cells, CD4 − CD8 + . (H) Same as (G) , but for CD4 + CD8 − T cells. (I) ELISA for IFNg in supernatants from cocultures as described in (F) . For (G–I) , mean and SEM are represented, n = 5–7, pooled from three independent experiments. **pv<0.01 for two-way ANOVA with Šidák’s multiple comparisons tests.

Article Snippet: To detect engineering via E6 target epitopes of the respective binders, cells were incubated at 0.5 μM in Cell Staining Buffer (Biolegend) with biotinylated peptides or biotinylated recombinant E6 protein (R&D Systems) after staining for Zombie L/D (Biolegend), washed and stained with streptavidin (Biolegend) and other antibodies for phenotypic characterization.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Reverse Transcription, Binding Assay, Amplification, Nucleic Acid Electrophoresis, Control, Molecular Weight, Isolation, Flow Cytometry, Incubation