Article Figures & Data
Figures
- Figure 1.
Antitumor response and improved survival with erdafitinib and anti–PD-1 combination. A, Efficacy study design in FGFR2K660N;p53mut (FKNP) lung tumor–bearing mice. Mice were treated with either control (vehicle + rat IgG2a isotype), anti–PD-1, erdafitinib, or erdafitinib + anti–PD-1, and were monitored for survival after 4 weeks of dosing (n = 8/group). wks, weeks. B, Representative serial MRIs of lung tumors in FKNP mice treated for 4 weeks. Images represent baseline and 2, 4, and 6 weeks after the start of treatment (red H, the heart). C, Percentage of tumor volume changes in each treatment group quantified from MRI using 3D Slicer software at baseline and 2, 4, and 6 weeks after the start of treatment. Solid black line represents the treatment duration. D, Kaplan–Meier survival curves across treatment groups in FKNP lung tumor–bearing mice, demonstrating significant survival benefit with combination over either control (a, P < 0.0005) or erdafitinib monotherapy (b, P < 0.004, log-rank test). Solid black line represents the treatment duration.
- Figure 2.
Inhibition of FGFR signaling in FKNP tumors. A, Pharmacodynamic study design. Pretreatment blood was collected a day before the start of the treatment. Treated mice were harvested for tumors and blood on day 8 for IHC, flow cytometry, and TCR sequencing analyses. Control n = 23, anti–PD-1 n = 20, erdafitinib n = 21, and combination n = 24. BID, every day; h, hours; QOD, every other day. B, Representative H&E sections of tumor-bearing lungs at day 8 of treatment for each group. Scale bar, 1 mm. C, Changes in percentage of tumor volume of individual mice in each treatment group after a week of treatment, quantified from MRI. ****, P < 0.0001, one-way ANOVA. Changes in expression of pFRS2 (D) and pS6 (E) in treatment groups were quantified using FFPE lung sections. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, Welch t test. Representative IHC images for pFRS2 (F) and pS6 (G). Scale bar, 50 μm.
- Figure 3.
Effects of erdafitinib and anti–PD-1 on T-cell infiltration and proliferation in FKNP tumors. Changes in immune cell infiltration and proliferation in FKNP tumor-bearing lungs at day 8 of treatment were analyzed. A and B, Representative IHC images (left) and quantified changes (right) by treatment are shown for Ki67+ (A) and CD3+ (B). Control n = 9, anti–PD-1 n = 5, erdafitinib n = 6, and combination n = 9. C–J, Flow cytometry analyses are shown for proliferative epithelial cells (EpCAM+Ki67+; C), T cells (CD3+; D), proliferative T cells (CD3+Ki67+; E), CD8+ cytotoxic T cells (CD8+; F), CD4+ helper T cells (CD4+; G), CD8+ central memory (CD8+CD62L+CD44+; H), and CD8+ effectors (CD8+CD62L−CD44+; I). Control n = 14, anti–PD-1 n = 13, erdafitinib n = 12, and combination n = 14. For A–I: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Welch t test. J, Changes in infiltrating CD8+ and CD4+ cells are stratified by tumor response. Boxplots show minimum value, 25th percentile, median, 75th percentile, and maximum values. Responders: >30% tumor regression. **, P = 0.0012; ***, P = 0.0001, two-tailed Wilcoxon rank sum test.
- Figure 4.
Changes in immune cell infiltration and T-cell exhaustion with treatment. Flow cytometry analyses of TILs in FKNP tumor–bearing lungs at day 8 of treatment. Changes with treatment in TAMs (CD11c+CD11b−; A) and proliferative TAMs (CD11c+CD11b−Ki67+; B). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Welch t test. C, Changes with treatment in Tregs (CD4+Foxp3+CD25+). Changes with treatment in triple-positive exhaustion markers in CD8+ (CD8+PD-1+TIM3+LAG3+; D) and CD4+ (CD4+PD-1+ TIM3+LAG3+; E) T cells. *, P < 0.05; **, P < 0.01, Welch t test. F, Association of abundance of NK cells and TAMs with tumor response. Boxplots show minimum value, 25th percentile, median, 75th percentile, and maximum values. Responders: >30% tumor regression. **, P < 0.001; ***, P < 0.0005, two-tailed Wilcoxon rank sum test.
- Figure 5.
Erdafitinib inhibits PD-L1 expression. Changes in PD-L1 expression in FKNP tumor–bearing lungs at day 8 of treatment were analyzed. Representative IHC images by treatment (A) and quantified changes by treatment (B). Scale bar, 200 μm. Flow cytometry showing the frequency of PD-L1+EpCAM+ cells (C) and PD-L1+TAMs (D). PD-L1 expression was assessed in archived human lung cancer patient samples with FGFR alterations or KRAS mutations by IHC (E), with images representative of a wide range of PD-L1 expression (scale bar, 200 μm for FGFR and 400 μm for KRAS), and PD-L1 H-score (F), plotted vs. percentage of CD3 positivity per sample for patients with FGFR (n = 6) or KRAS (n = 83) alterations. Kato III cells (FGFR2-amplified human gastric cancer model) were treated with 0 to 500 nmol/L erdafitinib as indicated in the presence of IFNγ (5 ng/mL), and percentage decrease in PD-L1 expression relative to vehicle control–treated cells was assessed 24 hours later by flow cytometry (G) and FGFR signaling (H) evaluated 1.5 hours following treatment by Western blot. Samples cultured in the presence or absence of 5 ng/mL IFNγ included; tubulin probed as protein loading control. h, hours; NA, not applicable. **, P < 0.01; Welch t test.
- Figure 6.
Erdafitinib and anti–PD-1 treatment alter T-cell infiltration and clonality. A, T-cell infiltration and T-cell clonality were determined by TCRβ immunosequencing of FKNP lung tumors harvested at day 8 posttreatment start. Mean with SEM is shown for each group. Control n = 10, anti–PD-1 n = 8, erdafitinib n = 9, and combination treated n = 9. T-cell fraction (B) and clonality (C) in lung tumors in responders (>30% decrease in tumor volume) and nonresponders. D, Number of expanding T-cell clones in peripheral blood at day 8 compared with baseline, which are also found in tumors, are depicted for each treatment group (P = 0.139, Mann–Whitney). E, Fraction of expanded T-cell clones in peripheral blood at day 8 that were below detection at baseline are shown for individual treatment groups (post hoc P = 0.041). Boxplots show minimum value, 25th percentile, median, 75th percentile, and maximum values. Tx, treatment. *, P < 0.05; **, P < 0.001, two-tailed Wilcoxon rank sum test.
Additional Files
Supplementary Data
- Supplementary Figure Legends, Tables and Methods - Supplemental figure legends 1-8, Supplementary tables 1-5, and Supplementary methods
- Supplemental Figures 1-8 - Supplemental Figures 1-8
Volume 7, Issue 9
