Fig 2: aPD1-IL18mut induces tumor cell killing and IFN-γ production in human TILs. (A) Cytokine profiling of ex vivo stimulated TILs. Heatmap illustrating z-scored means of 35 detected soluble cytokines in the supernatants of human cancer digests incubated with 200 nM of either isotype, aPD1, Iso-IL18mut, aPD1+Iso-IL18mut or aPD1-IL18mut + IL-12 (100 pg/mL) for 48 hours. Replicate numbers differ between treatment groups: Isotype, aPD1, and aPD1-IL18mut each n=8 donors; Iso-IL18mut and aPD1+Iso-IL18mut each n=4 donors. (B) Box plots showing fold-change to isotype in absolute soluble cytokine secretion. Statistical testing (mixed-effects model with Benjamini-Hochberg correction) was performed pairwise relative to each donor’s isotype control. Only cytokines with p<0.05 across donors are displayed. For CXCL9 and IFN-γ, all replicates were within quantification range; CXCL10 values were only available for donors within the quantifiable range (isotype n=7, aPD1 n=7, Iso-IL18mut n=2, aPD1+Iso-IL18mut n=3, aPD1-IL18mut n=4). Non-quantifiable values (above upper or below lower limit of quantification) were excluded by the platform. A table with donor-level values is provided in online supplemental table 4. (C) Box plots showing absolute IFN-γ expression was compared between TILs receiving IL-18 challenge. (D) Schematic representation of TICS assay setup incubated for 48 hours. (E) Box plots representing tumor cell survival relative to isotype. (F) IFN-γ secretion measured by ELISA as fold change to isotype. (G) %IFN-γ+ of (left) CD44+ Tconv or (right) CD44+ CD8+ T cells acquired by flow cytometry relative to isotype. Box plots indicate the median, 25th to 75th percentiles, minimum/maximum and dots represent individual donors. Data were pooled from two individual experiments (n=8 biological replicates total). (E–G) Statistical analyses were performed using a nonparametric Friedman test with multiple comparisons to aPD1-IL18mut condition. *p<0.05, **p<0.01, ***p<0.001. aPD1-IL18mut, anti-PD-1-interleukin-18 mutein immunoconjugate; IFN, interferon; IL-18, interleukin-18; PD-1, programmed cell death protein 1; Tconv, conventional CD4+ T cell; TICS, tumor-immune co-culture system; TILs, tumor-infiltrating lymphocytes.

Fig 3: Mechanistic effects of PDA‐CV@PI combined with IRE for HCC treatment. A) CD45+CD3+CD4+; B) CD45+CD3+CD8+; C) CD45+CD3+CD8+PD‐1+; D) CD4+CD25+Foxp3+; E) WB results of PD‐1 and related pathway proteins; and F) statistical bar chart of PD‐1 and related pathway proteins by WB. Values are expressed as mean ± SD. (versus control group, *: p < 0.05, **: p < 0.01, and ***: p < 0.001 determined by Student's t‐test).

Fig 4: aPD1-IL18mut induces CD4+ T cell-driven tumor control through activation of Th1-like effector cells in YUMM1.7 tumors. (A) Schematic representation of YUMM1.7 experimental study. hPD-1 mice were injected s.c. with YUMM1.7 tumor cells and treated intraperitoneally with aPD1-IL18mut or respective controls at day 9 and 13 post-tumor cell injection. (B) Tumor volume (mean+SEM) of hPD-1 mice with subcutaneous YUMM1.7 tumors treated with corresponding treatments. n=5–6 animals per treatment group. Statistical analysis was performed using two-way ANOVA with multiple comparisons with Tukey correction. (C) Tumor volume (mean+SEM) for depletion experiment comparing isotype- and aPD1-IL18mut-treated mice given corresponding depletion antibodies or isotype control (n/a). Isotype and aPD1-IL18mut were administered on day 9 and 13 post tumor cell injection. Depletion antibodies were administered at day 7 post tumor cell injection, 1 day following each treatment administration and then two times per week. n=5 or 6 mice per treatment group. (D) Representative contour plots gated on CD4+FOXP3− Tconv showing PD-1 against IL-18Rɑ of different organs on day 3 after isotype treatment. (E) Box plots showing the frequency of PD-1+IL-18Rɑ+ among CD4+FOXP3− Tconv across different organs. (F) Box plots representing PD-1 and IL-18Rɑ frequencies of intratumoral CD44+ CD4+ Tconv. (G–I) Representative histograms and box plots depicting mean fluorescence intensity (GeoMean) of (G) T-bet, (H) CXCR6 and (I) IFN-γ (stained after 4 hours of PMA/ionomycin restimulation; brefeldin A and monensin were added after 1 hour) across different PD-1 and IL-18Rɑ populations of CD44+ CD4+ Tconv. (J–O) Mice were treated with either isotype control (10 mg/kg), aPD1 (10 mg/kg) + Iso-IL18mut (1 mg/kg) or aPD1-IL18mut (1 mg/kg) and organs harvested at day 3 post administration. Box plots depicting the frequency of (J) CD69, (K) CD25, (L) Ki67, (M) IFN-γ and (N) GzmB among CD44+ PD-1+IL-18Rɑ+ CD4+ Tconv across different treatment groups. (O) Intratumoral IFN-γ titers of snap-frozen and homogenized tumor fragments across treatment groups. Box plots indicate the median, 25th to 75th percentiles, minimum/maximum and dots represent individual mice. For comparisons between three or more groups, a parametric one-way ANOVA with multiple comparisons with Tukey correction was used. In (O) a non-parametric one-way ANOVA with multiple comparisons was used. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; aPD1-IL18mut, anti-PD-1-interleukin-18 mutein immunoconjugate; dLN, draining lymph node; GzmB, granzyme B; hPD-1, human PD-1; IFN, interferon; IL-18R, interleukin-18 receptor; PD-1, programmed cell death protein 1; PMA, phorbol 12-myristate 13-acetate; s.c., subcutaneously; Tconv, conventional CD4+ T cell.

Fig 5: aPD1-IL18mut induces CD8+ T cell-driven tumor control accompanied by localized Th1 cytokine responses in MC38 tumors. (A) Tumor growth for indicated treatments. hPD-1 mice were treated intravenously with indicated treatments at day 10 and 17 post tumor cell injection (corresponds to day 0 and 7 post-treatment). Data show means+SEM for n=9 mice per group. Statistical analysis was performed using two-way analysis of variance with multiple comparisons with Tukey correction. (B) Kaplan-Meier survival curves of mice treated as indicated, showing n=9 mice per group. P values were computed using the log-rank test comparing each treatment group to the aPD1-IL18mut-treated group. (C) Schematic outline of immuno-pharmacodynamics study. MC38-bearing hPD-1 mice were treated with either vehicle or aPD1-IL18mut at an approximate tumor size of 400 mm3 and tumors or blood were harvested at selected time points post-treatment. (D) Frequencies of intratumoral CD3+ T cells among live CD45+ cells across time post treatment with vehicle (black) or aPD1-IL18mut (teal). Shown are means+SEM of three to six mice per group. (E) Count of CD45+ cells in 500 µL peripheral blood across time post-treatment with vehicle (black) or aPD1-IL18mut (teal). Shown are means+SEM of three to six mice per group. (F) Expression (GeoMean) of (left) PD-1 or (middle) IL-18Rɑ plotted for indicated cell populations. (right) Frequency of PD-1+IL-18Rɑ+ cells among indicated cell populations. Data are shown for vehicle-treated animals (pool of day 2 and 7 post-treatment). Dots represent individual mice, horizontal lines indicate the mean. (G) Counts (per 100 µL of blood and 0.1 g of tumor) of indicated cell populations of vehicle-treated or aPD1-IL18mut-treated animals at day 5 post treatment. Dots indicate individual mice, horizontal lines indicate the mean. (H) Box plots indicating frequencies of intratumoral (left) CD44+ or (right) naive CD8+ T cells. Data represent five to six animals per group. (I) Frequencies of intratumoral (left) CD44+ or (right) naïve CD4+ T cells. CD4+ T cells in this figure represent the total CD4+ T-cell pool. Box plots indicate the median, 25th to 75th percentiles, minimum/maximum and dots represent individual mice. Statistical analysis in (G–I) was performed using an unpaired t-test. (J) Tumor growth curves of vehicle-treated versus aPD1-IL18mut-treated animals with depletion antibodies. Depletion antibodies were administered two times per week from day 2 before vehicle or aPD1-IL18mut treatment. n=7 mice per group, data are shown as means+SEM. (K–M) Peripheral (blood) or intratumoral cytokine titers for (K) IFN-γ, (L) TNF-ɑ and (M) GM-CSF measured at indicated time points. Data indicate means+SEM of one to six mice per group. Non-quantifiable values (above upper or below lower limit of quantification) were excluded. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. aPD1-IL18mut, anti-PD-1-interleukin-18 mutein immunoconjugate; GM-CSF, granulocyte-macrophage colony-stimulating factor; hPD-1, human PD-1; IFN, interferon; IL-18R, interleukin-18 receptor; NK, natural killer; PD-1, programmed cell death protein 1; s.c., subcutaneously; TNF, tumor necrosis factor.