Figure 1 from Preclinical Characterization and Phase I Trial Results of a Bispecific Antibody Targeting PD-L1 and 4-1BB (GEN1046) in Patients with Advanced Refractory Solid Tumors

Abstract

<p>GEN1046 induces dose-dependent, conditional T-cell proliferation and cytokine production and enhances antigen-specific T-cell–mediated cytotoxicity <i>in vitro</i>. <b>A</b> and <b>B,</b> Activated T cells were cocultured with autologous iDCs in the presence of GEN1046 or control antibodies (0.125 µg/mL), and T-cell/iDC clusters were visualized over time by live-cell imaging. Quantification of the number of T cells in contact with a given DC on average (<b>A</b>), or over time (<b>B</b>; left), as well as the duration of these DC/T-cell clusters (<b>B</b>; right) is shown. ****, <i>P</i> < 0.0001; ns, not significant; Mann–Whitney <i>U</i> test. <b>C,</b> Induction of 4-1BB signaling by GEN1046 or control antibodies was assessed using a 4-1BB reporter assay. <b>D,</b> Blockade of the PD-1/PD-L1 interaction by GEN1046 or control antibodies was assessed using a PD-1/PD-L1 blockade bioassay. <b>E,</b> CFSE-labeled human PBMCs were stimulated with anti-CD3 (0.1 µg/mL) and incubated with GEN1046 or control antibodies (0.2 µg/mL) for 4 days. CFSE dilution in CD8<sup>+</sup> T cells was analyzed by flow cytometry, and the expansion index was calculated. Data shown are the fold change in expansion index of treatment groups, relative to untreated cells of individual donors, as well as mean ± standard deviation (<i>n</i> = 11). ****, <i>P</i> < 0.0001; **, <i>P</i> < 0.01; *, <i>P</i> < 0.05, Friedman test with Dunn multiple comparisons test. <b>F,</b> CD8<sup>+</sup> T cells were electroporated with RNA encoding a CLDN6-specific TCR and PD-1, labeled with CFSE, and cocultured with autologous DCs electroporated with CLDN6-encoding RNA in the presence of GEN1046 or control antibodies (0.2 µg/mL) for 4 days. Proliferation was measured by CFSE dilution as described in <b>E</b>. ****, <i>P</i> < 0.0001, Friedman test with Dunn multiple comparisons test. <b>G,</b> IFNγ concentrations in supernatant taken after 48 hours from cultures as described in <b>F</b>. Data shown are mean concentration ± standard deviation of triplicate wells from one representative donor (<i>n</i> = 3 donors). <b>H–K,</b> CD8<sup>+</sup> T cells were electroporated with RNA encoding a CLDN6-specific TCR and were preactivated for 24 hours in coculture with CLDN6-expressing MDA-MB-231 cells (MDA-MB-231 hCLDN6) to induce 4-1BB expression. Subsequently, the preactivated CD8<sup>+</sup> T cells were transferred to cocultures with previously seeded MDA-MB-231 hCLDN6 tumor cells (hCLDN6<sup>+</sup> PD-L1<sup>+</sup>) in the presence of GEN1046 or control antibodies (0.2 µg/mL). <b>H</b>–<b>I,</b> After 48 hours, expression of cytotoxic mediator GZMB and degranulation marker CD107a by the CD8<sup>+</sup> T cells was assessed; pooled data from four experiments (<i>n</i> = 3–12) are depicted. MFIs are expressed as relative values compared with the isotype control condition. <b>J,</b> Cytotoxicity of the CD8<sup>+</sup> T cells toward MDA-MB-231 CLDN6 cells was monitored by electrical impedance measurement over 5 days using the xCELLigence real-time cell analysis. Representative data (mean ± standard deviation of triplicate wells) from cocultures derived from one individual donor are shown (<i>n</i> = 12). Data were normalized to the time point of coculture start and expressed relative to tumor-cell cultures without T cells (without T cells set to 100%). <b>K,</b> AUC (total area) analysis of cytotoxicity data (<i>n</i> = 11–12). ****, <i>P</i> < 0.0001; ***, <i>P</i> < 0.001; **, <i>P</i> < 0.01; *, <i>P</i> < 0.05, mixed-effect analysis with Dunnett multiple comparisons test. MFI, median fluorescence intensity; ns, not significant.</p>

Related Publications