anti her3  (R&D Systems)

Journal: Frontiers in Oncology

Article Title: Antitumor activity of PAbs generated by immunization with a novel HER3-targeting protein-based vaccine candidate in preclinical models

doi: 10.3389/fonc.2024.1472607

Figure Lengend Snippet: Polyclonal antibody response of Mv-HER3. (A) C57BL/6 mice were immunized with HER3-ECD (200 μg) adjuvanted in VSSP (200 μg)/Montanide (intramuscularly (im.), 50 μL). Four doses were administered every 14 days. Blood samples were drawn in specified days, and (B) anti-HER3-induced PAbs were titrated by ELISA. (C) Recognition of related HER receptors and a His-tag-irrelevant protein (PDL1-His) by immune sera (diluted 1:1000) was evaluated using ELISA. The preimmune (PI) sera (dotted red line) served as an additional specificity control. (D) Recognition of the four subdomains of HER3-ECD by immune sera was determined by ELISA. In the graphs, the dotted red line represents the average of optical density against all four subdomains for the PI sera. (E) Recognition of HER3-positive (SKBR3, DU145, PC9ER, LNCap) and HER3-negative (PC3, negative control) human tumor cells by immune sera (1:200, yellow histograms) was determined by flow cytometry. Cells incubated with the preimmune sera (PI, blue histograms) or stained directly with the conjugate (red histograms) served as technical controls. All results shown are representative of at least two experiments performed individually.

Article Snippet: After blocking, membranes were incubated overnight with primary antibodies: anti-HER3 (AF234, R&D Systems, USA), anti-HER2 (AF1129, R&D Systems), anti-HER1 (AF231, R&D Systems), or ß-actin (MAB8929, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Negative Control, Flow Cytometry, Incubation, Staining

Journal: Frontiers in Oncology

Article Title: Antitumor activity of PAbs generated by immunization with a novel HER3-targeting protein-based vaccine candidate in preclinical models

doi: 10.3389/fonc.2024.1472607

Figure Lengend Snippet: Antitumor effect of vaccination with the HER3-based vaccine candidate in the murine 3LL-R3 tumor model. (A) Anti-HER3 PAbs induced were titrated using ELISA against mErbB3-ECD. (B) Recognition of mErbB3 on 3LL-R3 tumor cells by anti-HER3 PAbs present in the immune sera (1:200, yellow histograms) was analyzed by flow cytometry. Cells incubated with preimmune sera (PI, blue histograms) or stained directly with the conjugate (red histograms) served as controls. (C) 3LL-R3 cells were treated with immune sera (heat inactivated and 1:20 diluted). After 96 h, cell viability was determined using the MTT assay. PI sera and doxorubicin (Dx, 10 μM) served as negative and positive controls, respectively. Differences among treatment means in a representative experiment were analyzed by one-way ANOVA, with Tukey’s test applied for multiple comparisons. (D) Mice immunized as described were challenged intravenously with 50,000 3LL-R3 cells. The image displays lungs extracted from immunized or control mice 3 weeks later. Scale bar, 1 cm. (E) Media ± SD of tumor weights of five animals per group is shown and compared using a t -test. Significant differences are represented as *** p < 0.001 and **** p < 0.0001. All results shown are representative of at least two experiments performed individually.

Article Snippet: After blocking, membranes were incubated overnight with primary antibodies: anti-HER3 (AF234, R&D Systems, USA), anti-HER2 (AF1129, R&D Systems), anti-HER1 (AF231, R&D Systems), or ß-actin (MAB8929, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Incubation, Staining, MTT Assay, Control

Journal: Frontiers in Oncology

Article Title: Antitumor activity of PAbs generated by immunization with a novel HER3-targeting protein-based vaccine candidate in preclinical models

doi: 10.3389/fonc.2024.1472607

Figure Lengend Snippet: HER receptor degradation was promoted by the sera obtained from mice vaccinated with the Mv-HER3 candidate. SKBR3, DU145, PC9ER, LNCap, H1975, and PC9 cell lines were incubated for 24 h with a mixture of anti-HER3 sera (1:100 diluted). Cells treated with a mixture of preimmune (PI) sera (1:100 diluted) served as a control. Protein expression of HER3 was determined by Western blotting (left panel). In these experiments, β-actin was used as a loading control. In DU145 and LNCap cells, HER2 and HER1 expression was also analyzed by Western blotting after 24 h of treatment with immune sera (1:100 diluted) (right panel). The images display autoradiography films corresponding to the detection of the molecules above mentioned in one representative experiment of two conducted for each cell line.

Article Snippet: After blocking, membranes were incubated overnight with primary antibodies: anti-HER3 (AF234, R&D Systems, USA), anti-HER2 (AF1129, R&D Systems), anti-HER1 (AF231, R&D Systems), or ß-actin (MAB8929, R&D Systems).

Techniques: Incubation, Control, Expressing, Western Blot, Autoradiography

Journal: Frontiers in Oncology

Article Title: Antitumor activity of PAbs generated by immunization with a novel HER3-targeting protein-based vaccine candidate in preclinical models

doi: 10.3389/fonc.2024.1472607

Figure Lengend Snippet: Inhibition of cell viability in a panel of human tumor cell lines treated with sera obtained from mice immunized with the Mv-HER3 candidate. (A) Cells from the SKBR3, DU145, PC9ER, H1975, LNCap, and PC9 cell lines were treated for 96 h with a mixture of sera obtained from mice immunized with the Mv-HER3 vaccine candidate (heat-inactivated and diluted 1:20). Cell viability was quantified using the MTT assay. Preimmune (PI) sera served as a negative control. TKI specific for wild-type (AG1478) or T790M-mutant HER1 (osimertinib) were used as positive controls of cytotoxicity induction as follows: 10 µM of AG1478 (for SKBR3, DU145, LN-Cap, and PC9 cells) or 1 µM osimertinib (for PC9ER and H1975 cells). The graphs represent one experiment that is representative of at least two conducted for each tumor cell line. Differences among means were analyzed using one-way ANOVA, with the Tukey’s test applied for multiple comparisons. Significant differences among treatments are represented as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (B) Caspase 3 activation and (C) DNA fragmentation in human H1975 tumor cells treated with purified Pabs from rabbits immunized with the Mv-HER3 vaccine candidate. Untreated cells and cells treated with irrelevant Pabs served as negative controls, while MitC (5 μg/mL) was used as a positive control for both assays. The results shown are representative of two independently performed experiments.

Article Snippet: After blocking, membranes were incubated overnight with primary antibodies: anti-HER3 (AF234, R&D Systems, USA), anti-HER2 (AF1129, R&D Systems), anti-HER1 (AF231, R&D Systems), or ß-actin (MAB8929, R&D Systems).

Techniques: Inhibition, MTT Assay, Negative Control, Mutagenesis, Activation Assay, Purification, Positive Control

Journal: Frontiers in Oncology

Article Title: Antitumor activity of PAbs generated by immunization with a novel HER3-targeting protein-based vaccine candidate in preclinical models

doi: 10.3389/fonc.2024.1472607

Figure Lengend Snippet: Antitumor effect of PAbs generated by immunization with the HER3-ECD-specific vaccine candidate. (A) The experimental scheme followed: athymic mice were challenged subcutaneously with 3 * 10 6 cells of DU145 tumor cell line (diluted in 100 µL of PBS and injected into the right flank). Once tumors were palpable, mice were treated intraperitoneally (IP) with 1 mg of Mv-HER3-induced PAbs (from immunized rabbits). (B) Five doses were administered, and tumor growth kinetic curves were determined. (C) Average weight and (D) images of tumors extracted from mice treated with Mv-HER3-induced or irrelevant PAbs are shown. Scale bar, 1 cm. Tumor growth kinetic curves were compared using two-way ANOVA, while differences in means for average weight in (C) were analyzed using a t -test. Significant differences are represented as ** p < 0.01; **** p < 0.0001. The results shown are representative of two independently performed experiments.

Article Snippet: After blocking, membranes were incubated overnight with primary antibodies: anti-HER3 (AF234, R&D Systems, USA), anti-HER2 (AF1129, R&D Systems), anti-HER1 (AF231, R&D Systems), or ß-actin (MAB8929, R&D Systems).

Techniques: Generated, Injection