Fig 2: Intratumorally-injected reovirus sensitizes local and distant cold tumors for subsequent treatment with CD3xTRP1 therapy. (A) Expression of activation markers on splenic CD8+ or CD4+ T cells 5 days after reovirus or phosphate-buffered saline (PBS) administration (n=5/group). (B) Treatment schedule of experiment described in (C–G). Mice (n=8–10/group) were subcutaneously inoculated with KPC3.TRP1 cells in the right flank. Seven days later, another KPC3.TRP1 or KPC3 tumor was inoculated in the left flank. Mice received intratumoral (i.t.) injections of reovirus (107 plaque-forming units (pfu)) in the primary right tumor. Then, mice were sacrificed for intratumoral analysis at 3 and 7 days post reovirus injections (p.i.) (panels C–F; pooled results of two independent experiments with similar results) or mice received subsequent intraperitoneal (i.p.) injections of 12.5 µg CD3xTRP1 bispecific antibodies (bsAbs) and tumor size was monitored (panels G and H). (C) Reovirus genomic segment 4 (S4) copy numbers in local and distant tumors 3 days after reovirus treatment or PBS as control. (D) S4 copy numbers present in other organs of reovirus-treated mice 3 days after reovirus administration. Dashed horizontal line represents average S4 copy numbers in PBS-treated mice. (E) Heatmap of relative expression of interferon response genes in local and distant tumor as determined by quantitative reverse transcription PCR. Numbers indicate fold change versus PBS-treated local tumors. (F) Frequency of T cells in local and distant tumors of mice 7 days after local reovirus administration. Data presented as mean±SEM. Differences between PBS and reovirus in panels A and C–F were determined with unpaired t tests. (G) Individual tumor growth curves of local and distant tumors of mice (n=8/group) treated with CD3xTRP1. Dashed red lines indicate timing of injection. (H) Individual tumor growth curves of local and distant TRP1-expressing (KPC3.TRP1) or wild-type (WT) KPC3 tumors of mice after i.t. treatment with reovirus and subsequent i.p. injections with CD3xTRP1. Dashed lines indicate timing of injection with reovirus (blue) or CD3xTRP1 (red). Significance levels: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. TDLN, tumor-draining lymph node.

Fig 3: Combination treatment of reovirus and CD3xTRP1 bispecific antibodies (bsAbs) induces regression of established TRP1-expressing tumors. (A) Design of experiment described in (B–F). Mice (n=8–10/group) with established KPC3.TRP1 tumors were intratumorally (i.t.) injected with reovirus (107 plaque-forming units (pfu)) on 3 consecutive days. After 4 days, mice received intraperitoneal (i.p.) injections of 12.5 µg CD3xTRP1 bsAbs (CD3xTRP1) or phosphate-buffered saline (PBS) as control. (B) Average tumor growth curves±SEM. Dashed lines indicate timing of injection with reovirus (blue) or CD3xTRP1 (red). Differences in mean tumor volumes versus reovirus+CD3xTPR1 treatment on day 21 are determined by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. (C) Relative changes in tumor volume of individual mice from the start of CD3xTRP1 bsAb treatment. Indicated is the number of mice with tumor regressions. (D) Kaplan-Meier survival graphs of mice in indicated treatment groups. (E) Overlayed dot plots indicating percentage of TRP1+ cells on representative tumors of each group. Black dots show background staining of secondary antibody. Red dots show staining using primary aTRP1 antibody followed by the secondary antibody. (F) Quantification of TRP1 expression. Dashed line indicates mean background staining of secondary antibody. Data represent mean±SEM. (G) Design of experiment described in (H–J). Mice (n=8–10/group) with established B16.F10 tumors were i.t. injected with reovirus (107 pfu) on 3 consecutive days. After 4 days, mice received i.p. injections of 12.5 µg CD3xTRP1 bsAbs or PBS as control. (H) Individual growth curves of B16.F10-bearing mice receiving indicated treatments. (I) Relative changes in tumor volume from start of CD3xTRP1 bsAb treatment. Indicated is the number of mice with tumor regressions. (J) Kaplan-Meier survival graphs of mice in indicated treatment groups. Log-rank test was used to compare differences in survival. Significance levels: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Fig 4: TGF-ß inhibition diminishes the efficacy of Reo&CD3-bsAb therapy in the KPC3.TRP1 tumor model. A, Overview of the experiment described in B–G. Mice (n = 9–10/group) were subcutaneously engrafted with KPC3.TRP1 cells (1 × 105/mouse) and received TGF-ß-neutralizing antibodies (aTGF-ß, 200 µg/injection every 3 days) starting directly after tumor engraftment. Mice received Reo intravenously on days 14, 15, and 16 (108 pfu/injection) and received CD3-bsAbs intraperitoneally (12.5 µg/injection) on days 20, 22, and 24. Tumor growth was measured 3–5×/week. B, Individual tumor growth curves of mice receiving indicated treatments. C, Average tumor growth curves of mice receiving indicated treatments. One nonresponding mouse in the Reo&CD3-bsAbs group is excluded for clarity (see also B). Significant differences in average tumor growth were calculated on day 23. D, Relative changes in tumor volume of individual mice, calculated from the start of CD3-bsAb treatment. Indicated is the number of mice with tumor regressions in each group. E, Kaplan–Meier survival graphs of mice after indicated treatments. F, Quantification of TRP1 expression on CD45- cells within the end-stage KPC3.TRP1 tumors after indicated therapies. Gray values indicate corresponding background staining of secondary goat-anti-mouse antibody only. G, Correlation between TRP1 expression in tumors and the day of sacrifice. Data represent mean ± SEM. Significance in C was determined using an ordinary one-way ANOVA with Tukey multiple comparisons test. Log-rank tests were used to compare differences in survival in E. Significance levels: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. Figure A was created with BioRender.com.

Fig 5: TGF-ß blockade significantly enhances the efficacy of Reo&CD3-bsAb therapy in the MC38.TRP1 model of colon cancer. A, Overview of the experiment described in B–H. Mice (n = 9–10/group) were subcutaneously engrafted with MC38.TRP1 cells (5 × 105/mouse) and received TGF-ß-neutralizing antibodies (aTGF-ß, 200 µg/injection every 3 days) starting directly after tumor engraftment. Mice received Reo (intravenously, 108 pfu/injection) and CD3-bsAbs (intraperitoneally, 200 µg/injection) on days 14 and 16. Tumor growth was measured 3×/week. B, Individual tumor growth curves of mice receiving indicated treatments. C, Relative changes in tumor volume of individual mice from the start of CD3-bsAb treatment. D, Kaplan–Meier survival graphs of mice after indicated treatments. E, Frequency of nonresponders (NR), partial responders (PR; tumor regression/stagnation for more than 7 days), or complete responders (CR) within indicated treatment groups. F, Frequency of Rpl18+ and Reo µ1133–140 CD8+ T cells in the blood of mice after indicated treatments. G, Rechallenge experiment. All CR mice from D were subcutaneously engrafted with MC38.(TRP1) tumor cells (5 × 105/mouse) in the alternate flank on day 51 (MC38.TRP1) or day 85 (MC38) and tumor outgrowth was measured 3×/week. Indicated is the number of mice within each group that rejected the rechallenge. Data represent mean ± SEM. Log-rank tests were used to compare differences in survival in D. A ?2 test was used to determine statistical differences in response in E. Significance between groups in F was determined using an ordinary one-way ANOVA with Tukey multiple comparisons test. Significance levels: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. Figures A and G were created with BioRender.com.