Journal: Frontiers in Immunology

Article Title: A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors

doi: 10.3389/fimmu.2020.00895

Figure Lengend Snippet: Identification of the mimotopes of the anti-human programmed cell death 1 (hPD1) mAb Nivolumab and anti-mouse PD1 (mPD1) monoclonal antibody (mAb), and examination of their specificity. Colony blot assay was applied on clones of Escherichia coli individually expressing overlapping peptides spanning the entire extracellular domain of hPD1 (A) or mPD1 (B) with anti-hPD1 and anti-mPD1 mAbs used for detection, respectively, as described in the Materials and Methods section. In the colony blot assay with anti-hPD1 mAb, the detected clones are boxed with solid line. One positive clone with failed sequencing is boxed with a broken line. Capacity of the identified mimotopes JT–N1, JT–N2, and JT–N3 (C,D) and JT–mPD1 (E) with comparison to a control mimotope, in inhibiting the binding of anti-hPD1 and anti-mPD1 mAbs to recombinant hPD1 or mPD1 proteins, respectively, is shown. Recombinant hPD1 or mPD1 proteins were used for coating in a solid phase-based assay (ELISA), and binding of the respective mAbs to the coated proteins was evaluated alone or after preincubation with different examined concentrations of the respective mimotopes. The results are representative of at least two repeated experiments. Significant differences are indicated by asterisks (** P < 0.01, *** P < 0.001).

Article Snippet: Jurkat reporter cells expressing hPD1 or mPD1 were employed to examine the capacity of the mimotopes JT–N1 and JT–N2, and also JT–mPD1, respectively, in inhibiting the binding of the corresponding mAbs to the respective cells.

Techniques: Clone Assay, Expressing, Sequencing, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors

doi: 10.3389/fimmu.2020.00895

Figure Lengend Snippet: Examination of the specificity of the mimotopes JT–N1, JT–N2, and JT–mPD1 in cellular assays. (A) Jurkat T cells expressing high levels of hPD1 were used in a binding assay to examine the binding of anti-hPD1 mAb Nivolumab (50 ng/ml) alone or after preincubation with different concentrations of JT–N1, JT–N2, combination of both mimotopes, and a control mimotope. The inset shows reactivity of an antibody to hPD1 on Jurkat–hPD1 (gray histogram) and control Jurkat cells (open histogram) n = 4 for each data point. (B) Flow cytometry histograms of a representative experiment are shown. Open histogram, no Nivolumab; blue histogram, binding of Nivolumab alone; gray histograms, titration of JT–N1, JT–N2, combination of both mimotopes, and the control mimotope. (C) Jurkat T cells expressing mPD1 were used in a binding assay to examine the binding of anti-mPD1 mAb (10 ng/ml) alone or after preincubation with different concentrations of JT–mPD1 and the control mimotope. The inset shows reactivity of the anti-mPD1 mAb on Jurkat–mPD1 (gray histogram) and control Jurkat cells (open histogram) n = 3 for each data point. Significant differences are indicated by the P -values. (D) Flow cytometry histograms of a representative experiment are shown. Open histogram, no anti-mPD1 mAb; blue histogram, binding of the anti-mPD1 mAB; gray histograms, titration of the mimotope JT–mPD1, and the control mimotope. (E) Reporter gene (eGFP) expression of Jurkat hPD1 reporter cells activated by hPD–L1-expressing stimulator cells. The anti-hPD1 mAb (alone or after preincubation with JT–N1) was added as indicated n = 4 for each data point. The results are representative of at least two repeated experiments. Significant differences are indicated by asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Jurkat reporter cells expressing hPD1 or mPD1 were employed to examine the capacity of the mimotopes JT–N1 and JT–N2, and also JT–mPD1, respectively, in inhibiting the binding of the corresponding mAbs to the respective cells.

Techniques: Expressing, Binding Assay, Flow Cytometry, Titration