Supplementary Data from Targeted BiTE Expression by an Oncolytic Vector Augments Therapeutic Efficacy Against Solid Tumors

<p>Supplementary Methods, Supplementary Figures S1-S15 Fig. S1. Purification and binding specificity of MV-encoded BiTEs. (A) Purification of MV-expressed BiTEs. Vero cells were inoculated with MV-BiTE (MOI 0.03) and cell-free supernatant was harvested 62 h post inoculation. BiTEs were purified by immobilized metal affinity chromatography, washed and concentrated in centrifugal filter units in PBS. Representative Western blot of samples from designated steps of the purification process is shown (washing I/II: 10/20 mM imidazole; elution I/II: 300/500 mM imidazole). MV-expressed, purified BiTE (vpBiTE) was detected via N-terminal HA-tag. (B) Magnetic pull-down of BiTE-labeled cells. Tumor cells were incubated with vpBiTE. BiTE-labeled cells were purified via anti-HA-biotin antibody and anti-biotin magnetic beads. Western blot of column eluate (BiTE binding) and flow-through (unlabeled cells) with anti-β-actin antibody is shown. (C) Binding specificity. Binding of indicated vpBiTEs to recombinant protein was assessed by sandwich ELISA with rhCEA or non-relevant protein (rmPD-L1; rmCTLA-4). Data was normalized to background control (PBS) and is depicted as fold change over background. (D) Competitive binding assays. PBMCs (left panel) or tumor cells (right panel) were incubated with vpBiTEs. After incubation, remaining BiTEs were detected by ELISA of cell-free supernatants. Data was normalized to non-binding controls (PBS and MC38, respectively) and is depicted as fold change over nonbinding controls. (C, D) Mean values of triplicate samples with SD are shown. Statistical analysis was performed by one-way ANOVA, and p values were adjusted for multiple comparisons by Dunnett's test. rh/m: recombinant human/murine Fig. S2. MV-BiTE infection of murine tumor cells. (A, B) Replication kinetics and cytotoxic effects of MV-BiTEs in murine tumor cells. (A) MC38-CEA or (B) B16-CD20-CD46 cells were inoculated with indicated MV variants at MOI 1. Left panels: Viral progeny particles at designated time points were quantified in titration assays to generate one-step growth curves. Right panels: Cell viability was determined by XTT assay at indicated time points. (C, D) Infection of murine tumor cells with MV-BiTEs. (C) MC38-CEA or (D) B16-CD20-CD46 cells were inoculated with indicated MV-eGFP-BiTEs. Images were acquired 48 h post inoculation. Scale bars: 200 µm. (E) BiTE expression kinetics. Vero and MC38-CEA cells, respectively, were inoculated with MV-mCD3xhCEA at indicated MOIs. Supernatants were collected at designated time points and relative BiTE concentrations were determined by ELISA with rhCEA. Mean values of three replicates with SD are shown. Note that samples from Vero cells are identical to those presented in Fig. 1F. Fig. S3. Tumor growth curves for individual MC38-CEA-bearing mice. MC38-CEA-bearing C57BL/6J mice were subjected to mock, MV-mCD3xCD20 or MV-mCD3xCEA treatment. Tumor growth curves for individual mice are shown. The corresponding Kaplan-Meier survival analysis is shown in Fig. 3A. Fig. S4. Tumor growth curves for individual B16-CD20-CD46-bearing mice. C57BL/6J mice bearing subcutaneous B16-CD20-CD46 tumors received intratumoral injections of indicated viruses, carrier fluid (mock) or mCD3xCD20 vpBiTE, respectively. Tumor growth curves for individual mice are shown. Corresponding KaplanMeier survival plots are depicted in Fig. 3B and C. Fig. S5. Analysis of intratumoral MV-N and BiTE expression after MV-BiTE treatment of B16-CD20-CD46 tumors. C57BL/6J mice received injections of 1x106 cell infectious units of indicated MV-BiTE into established subcutaneous B16-CD20-CD46 tumors on five consecutive days. Mice were sacrificed and tumors were explanted directly after the first treatment (1st t.), directly after the fifth treatment (5th t.), 24 h post fifth treatment (p.t.), 48 h p.t. and 120 h p.t.. Tumor samples were subjected to reverse transcription quantitative PCR (RT-qPCR). Fold change expression of MV-N (A), and mCD3xCD20 and mCD3xCEA BiTEs (B) in MV-BiTE-treated tumors relative to mocktreated tumors is shown. Values were normalized to the expression of L13A. Mean values and SD are shown (n = 4 tumors for each time point, except n = 3 for 5th t. MV-mCD3xCEA and n = 1 for 120 h p.t. MV-mCD3xCD20). Fig. S6. Effects of UV inactivation on MV-BiTE efficacy. (A) Replication kinetics of UV-inactivated MV-BiTE. Vero cells were inoculated with MV-mCD3xCD20 at MOI 1 and viral progeny particles were titrated at indicated time points. pUV = partial UV inactivation; cUV = complete UV inactivation. (B) BiTE expression kinetics. B16-CD20-CD46 cells were inoculated with MV-mCD3xCD20 and relative BiTE concentrations in cell culture supernatants were determined by ELISA at designated time points. Individual samples are shown. (C) Efficacy of pUV MV-BiTE against melanoma in vivo. Established B16-CD20-CD46 tumors in immunocompetent mice were treated with intratumoral injections of carrier fluid (mock), MV-mCD3xCD20, or pUV MV-mCD3xCD20. Note that this is the same experiment as in Fig. 3B (i.e. mock and MV-mCD3xCD20 groups are identical). (D) Impact of complete UV inactivation on in vivo efficacy of unmodified MV. Immunocompetent C57BL/6J mice bearing established subcutaneous B16-CD20-CD46 tumors were treated by intratumoral injections of carrier fluid (mock), unmodified MV, or cUV MV. (C, D) Kaplan-Meier survival curves are shown with statistical analysis using log-rank (Mantel-Cox) test, and p values were corrected for multiple comparisons by the Bonferroni method. (E) Relative quantification of BiTE present in virus solution. BiTE binding to endogenous CD20 expressed by Granta cells was assessed by flow cytometry to determine the concentration of mCD3xCD20 vpBiTE corresponding to the amount of BiTE present in one dose of MV-mCD3xCD20. MFI - mean fluorescence intensity. Fig. S7. BiTE serum levels after MV-BiTE treatment in syngeneic mouse models. (A, B) Titration of purified BiTE and BiTE concentration in blood serum after MV-BiTE treatment. ELISA plates were coated with rhCEA and rhCD20, respectively. (A) mCD3xCEA and (B) mCD3xCD20 purified from MV-BiTE-infected cell supernatants were titrated in ten-fold serial dilutions. Blood was drawn from mice bearing (A) MC38-CEA and (B) B16-CD20-CD46 tumors of the indicated treatment groups at designated time points. Blood serum was tested for BiTE by ELISA (n = 3-5 mice per group and time point). Data are presented as fold change over background. Mean values with SD are shown. vpBiTE: virus-expressed and purified bispecific T cell engager; rh: recombinant human; p.t.: post fourth treatment. Fig. S8. Effects of MV vaccination on MV-BiTE efficacy. MV immunity was induced by prime-boost vaccination with intraperitoneal injections of highly purified oncolytic MV (MV-NIS) before B16-CD20-CD46 tumors were implanted subcutaneously. Established tumors were injected on five consecutive days with carrier fluid (mock), unmodified MV, or MV-BiTE as indicated. Kaplan-Meier survival curves are shown with statistical analysis using log-rank (Mantel-Cox) test; p values were corrected for multiple comparisons by the Bonferroni method. ns: not significant. Fig. S9. Tumor-infiltrating lymphocytes in immunocompetent mice after MV-BiTE therapy. (A, B) Flow cytometry of tumor-infiltrating lymphocytes. (A) MC38-CEA or (B) B16-CD20-CD46 cells were implanted subcutaneously into the flanks of C57BL/6J mice. Mice were treated with intratumoral injections of 1x10 6 ciu of MVBiTE according to the described treatment schedules. Tumors were explanted one day after the last treatment and tumor-infiltrating lymphocytes were analyzed by flow cytometry (n = 10 mice per group). (C) Intratumoral expression of T cell transcription factors. RNA was isolated from the B16-CD46-CD20 tumors described in (B) and subjected to RT-qPCR. mRNA levels of T cell differentiation-associated transcription factors FoxP3 and T-bet were assessed. Expression was normalized to L13A, and fold changes relative to the respective mean values of the mock group are shown. (A-C) Mean values with SD are shown. Statistical analysis was performed by one-way ANOVA, and p values were adjusted for multiple comparisons by Tukey's test. ns: not significant. Fig. S10. Differential expression of T cell-related genes after MV-BiTE treatment. (A) T cell activation genes. (B) T cell differentiation genes. (C) T cell proliferation genes. (D) Genes associated with T cell exhaustion and inhibition. (A-D) C57BL/6J mice bearing subcutaneous B16-CD20-CD46 tumors received intratumoral injections of carrier fluid (mock) or indicated viruses on four consecutive days. One day after the last treatment, mice were sacrificed, tumors were explanted and RNA was isolated and subjected to targeted transcriptome analysis. Note that the results shown in Fig. 3D and E were derived from the same experiment. Heat maps (A-C) were generated by unsupervised clustering using the advanced analysis package in nSolver 4.0 software after normalization to a set of internal reference genes and by scaling to give all genes equal variance. Heat map (D) was generated by agglomerative clustering using nSolver 4.0 software after normalization to the same set of reference genes. Yellow and blue indicate high and low expression, respectively. n = 5 tumors for mock, n = 8 tumors for MV-mCD3xCEA and MV-mCD3xCD20, respectively Fig. S11. MV-BiTE infection of tumor sphere cultures (TSCs). (A) BiTE binding to TSC cells expressing endogenous CEA. TSC cells were incubated with vpBiTEs and binding was detected by flow cytometry with an HA-specific antibody. (B) Replication kinetics. TSC8, TSC17 and TSC23 cells were inoculated with MV variants at MOI 1 and samples were collected at indicated time points. Progeny viral particles were quantified by titration assays to generate one-step growth curves. (C) BiTE expression kinetics. TSCs were inoculated with MV-hCD3xCEA at MOI 1. Supernatants were collected at indicated time points and relative BiTE concentrations were determined by ELISA with rhCEA. Individual samples are shown. MOI: multiplicity of infection; vpBiTE: virus-expressed and purified bispecific T cell engager; ciu: cell infectious units; rh: recombinant human. Fig. S12. BiTE serum levels after MV-BiTE treatment of patient-derived xenografts. (A) hCD3xCEA titration and BiTE concentration in blood serum after intratumoral MV-BiTE treatment. hCD3xCEA was titrated in ten-fold serial dilutions and analyzed by ELISA with rhCEA (left panel). Blood was drawn from TSC23 tumor-bearing NSG mice of the indicated treatment groups one day after the last treatment. Blood serum was tested for BiTE by ELISA with rhCEA (n = 5 mice per group) and compared to the amount of BiTE present in one treatment dose of MV-BiTE (right panel). (B) Analysis of BiTE serum levels after intravenous or intratumoral MV-BiTE injection. TSC8 tumor-bearing NSG mice received an intravenous (i.v.) or intratumoral (i.t.) injection of 1x106 ciu of MV-hCD3xCEA (n = 3 mice per group). Blood was drawn 15 min after MV-BiTE injection. Blood sera were analyzed by ELISA with rhCEA. (A, B) Data are presented as fold change over background. Mean values with SD are shown. rh: recombinant human. Fig. S13. Analysis of intratumoral MV-N and BiTE expression after MV-BiTE treatment of TSC8 tumors. Established subcutaneous TSC8 tumors were treated with intratumoral injections of 1x106 cell infectious units of MV-BiTE (MV-hCD3xCEA) on four consecutive days. On the first day of treatment, mice additionally received an intratumoral injection of 1x107 human PBMCs (n = 4 tumors per group). Tumors were explanted directly after the first treatment (t.), directly after the fourth t., 24 h post fourth treatment (p.t.), 192 h p.t. and 240 h p.t.. Tumor samples were subjected to reverse transcription quantitative PCR (RT-qPCR). Fold change expression of MV-N and hCD3xCEA in MV-BiTE-treated tumor samples relative to mock-treated tumor samples is shown. Values were normalized to the expression of β-actin. Mean values with SD are shown. Fig. S14. Tumor growth curves for individual TSC-bearing mice. TSC-bearing NSG mice were subjected to mock, PBMCs only, MV-hCD3xCEA only, or MV-hCD3xCEA + PBMCs treatment. Tumor growth curves for individual mice are shown. The corresponding Kaplan-Meier survival analyses are shown in Fig. 4C. Fig. S15. Analysis of intratumoral lymphocytes and CEA expression on tumor cells after MV-BiTE therapy. (A, B) Established subcutaneous TSC8 tumors were treated with intratumoral injections of 1x106 cell infectious units (ciu) of MV or MV-BiTE (MV-hCD3xCEA) on four consecutive days. On the first day of treatment, mice additionally received an intratumoral injection of 1x107 human PBMCs (n = 4 tumors per group). Tumors were explanted after the first treatment (day 6 post implantation), after the fourth treatment (day 9 post implantation) and eleven days post treatment (day 16 post implantation). (A) Persistence of CD3+ lymphocytes was analyzed by flow cytometry. Percentage of CD3 expressing cells of all live cells is shown. (B) CEA and EpCAM expression in tumor samples was analyzed by flow cytometry and compared to TSC8 tumor cells cultivated in vitro. The ratio of target tumor antigen (CEA) to non-target tumor antigen (EpCAM) expression is shown.</p>