Nature Medicine: doi: /nm.4356

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1 SUPPLEMENTARY FIGURES Supplementary Figure 1: Characterization of IVT mrna encoding highly active bsab. (a) IVT mrna quality and purity analysis on an Agilent 2100 Bioanalyzer. Full-length mrna peaks are highlighted in red. Marker peak and background signals are indicated in blue. (b) Western blotting and anti-6xhis-hrp detection of RiboMABs in supernatant of K-562 producer cells. Positive control, 20 ng of the corresponding purified recombinant protein; negative control, supernatant of mock electroporated K-562 cells. (c) Specific lysis induced by RiboMABs measured by luciferase-based cytotoxicity assay. Bulk human PBMCs and TAA-positive (solid line and symbols) or TAA-negative (dashed line/open symbols) luciferase transduced human tumor cell lines (E:T = 5:1) were co-cultured for 48 h with RiboMAB-containing supernatants of bi- (scfv) 2 mrna transfected K-562 cells (concentrations determined via ELISA). (d) Donor-dependent lysis of PA-1 cells by (top) RiboMAB-containing K-562 supernatant as compared to (bottom) purified recombinant protein bi-(scfv) 2. (e) CLDN6-positive PA-1 tumor cells electroporated with 20 µg ml -1 mrna encoding CD3xCLDN6 or an irrelevant bi-(scfv) 2 (control, PLAC1xCD3 directed against placenta specific 1) were co-cultured with human T cells (E:T = 5:1). Arrowhead, cluster of target and T cells recorded after 24 h. 1

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3 Supplementary Figure 2: Primarily hepatic targeting and translation of intravenously injected polymer/lipid formulated luciferase mrna. BALB/c mice were IV injected with 5 µg polymer/lipid formulated non-immunogenic luciferase mrna (n = 3) or with the polymer/lipid (TransIT) alone (n = 2). Luciferase activity was measured by bioluminescence imaging shortly after intraperitoneal injection of luciferin. Exposure time: 1 min. Sensitivity binning: 4 (6 h, 24 h) or 8 ( h). Red indicates high, blue low radiance signal. 3

4 Supplementary Figure 3: Sustained plasma levels of functional RiboMAB by repeated IV dosing of CD3xCLDN6 mrna. Biological activity of endogenously translated CD3xCLDN6 RiboMAB in plasma of immunodeficient NSG mice determined after IV administration (arrowheads) of polymer/lipid formulated mrna (5 µg) or of purified rcd3xcldn6 protein (200 µg kg -1 ). For ex vivo cytotoxicity assays RiboMAB-containing plasma (5%, technical triplicates) was analyzed in co-cultures of PA-1 cells and human PBMC (E:T = 5:1). Plasma harvest time points were 15 min (protein treated mice) or 30 min (mrna treated mice), 1 h, 6 h, 24 h, 72 h and 144 h after IV administration. ****P < (two-tailed Mann- Whitney U-test). 4

5 Supplementary Figure 4: Complete tumor elimination without systemic release of proinflammatory cytokines after IV treatment of mice with mrna encoding EpCAMxCD3 bi-(scfv) 2. NSG mice humanized with PBMC as effectors and bearing advanced subcutaneous (SC) tumor xenografts of EpCAMpositive OV-90 (mean tumor volume at start of treatment 100 mm 3 ) were treated with three doses of EpCAMxCD3 or luciferase mrna (3 µg polymer/lipid formulated mrna, n = 7 and n = 6, respectively) or with purified rcd3xcldn6 protein (200 µg kg -1, n = 6) or vehicle (n = 6) once per week. (a) Tumor growth kinetics for individual mice (top) (left mrna, right protein) and median of tumor growth per group (bottom). Significance determined by parametric two-way ANOVA (****P <0.0001, F value (DFn, DFd) = 39.6 (12, 137); ***P = , F value (DFn, DFd) = 3.2 (12, 139)). (b) Human proinflammatory cytokine profile of all NSG mice. Plasma harvested at day 17 (24 h "pre" treatment) and at day 26 (24 h post 2 nd treatment) were analyzed in cytokine ELISA (technical duplicates) and normalized to the vehicle control group. The median is shown. Significance of cytokine elevation was determined by unpaired two-tailed t test (****P <0.0001, t value = , DF = 24; *P = , t value = 2.4, DF = 19; ns, P = , t value = , DF = 13 26). 5

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7 Supplementary Figure 5: Lack of toxicity of polymer/lipid formulated non-immunogenic bi-(scfv) 2 mrna. (a) Plasma proinflammatory cytokine levels in human-pbmc-engrafted NSG mice treated IV with a single 3 µg dose of formulated non-immunogenic mrna coding for CD3xCLDN6 (n = 8) or luciferase (Luc, n = 8). 2 mg kg -1 OKT3 was administered as positive control for cytokine release (n = 4). Postinjection cytokine levels were normalized to the levels prior injection (dotted line). Scatter plots including medians are shown. Significance was determined by two-tailed Mann-Whitney U-test analysis of corresponding time points. (b) Liver enzymes (ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase) were measured in immunocompetent BALB/c mice IV injected four times (once per week) with DPBS, empty TransIT, 5 µg formulated Luc control, CD3xCLDN6 or EpCAMxCD3 mrna (n = 5 per group). Serum prior injection was from all groups (5 pools of 5 mice each). Boxes and whiskers with Min to Max and all sample points including medians are shown. Significance was determined by multiple t test with correction for multiple comparison using the Holm-Sidak method. (c) Plasma cytokine concentration (C p ) in immunocompetent BALB/c mice injected three times (once per week) with 5 µg non-immunogenic Luc control or CD3xCLDN6 mrna, with 5 µg immunogenic (unmodified Uridine instead of 1-methylpseudouridine, not HPLC-purified) CD3xCLDN6 mrna or with 200 µg kg -1 rcd3xcldn6 protein. The bi-(scfv) 2 was quantified by ELISA 6 h (mrna treated mice) or 15 min (protein treated mice) after the 1 st injection (n = 5 per group). Plasma IFN-α (n = 10) and TNF-α (n = 5) levels were measured 6 h after the third injection. Mean values of technical duplicates are shown. Lines indicate the medians. Significance (**P = , ***P < ) determined by twotailed Mann-Whitney U-test. 7

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