Photograph of an ex vivo tumorous kidney during a perfusion experiment. In this kidney, two renal arteries were connected to the perfusion system. During perfusion, the kidney was kept at 0-4 {degree sign}C and protected from light.

Overview of the results of perfusion experiment #2. High uptake of dual-labeled girentuximab was observed in two foci (*) of tumor thrombus in the renal vein (A, B). The central region of the tumor largely consists of necrosis, with little and inhomogeneous uptake of dual-labeled girentuximab. Histology confirms that the tumor center consists of fibrosis and necrosis (H&E staining detail, C), without CAIX expression (D).

PDF file - 41K, Clinical protocol and immunization schedule.

Supplementary Table 1 PDF file - 29K, Induration (mm) of the different injection sites in the DTH skin test

Photograph (A) and autoradiographic image (B) of the second control experiment showing high uptake of Iodine-131-girentuximab-IRDye800CW in tumor tissue. The low energy gamma radiation of Iodine-125 is not visible in this autoradiography.

PDF file - 92K, Immunological responses after DC vaccination.

Supplementary Figure 2 PDF file - 46K, Supplementary Figure 2 Plasmacytoid DCs acquire CCL21-driven chemotactic ability upon activation

Schematic overview of the different steps of the perfusion experiment.

Table S1. Overview of selected autophagy activating compounds, their structure, chemical and pharmacological classification and the supplier; Table S2. Significantly upregulated genes in all cell lines at all time points (fold change log2 {greater than or equal to} 1); Table S3. Significantly downregulated genes in all cell lines at all time points (fold change log2 {less than or equal to} -1); Table S4. Physicochemical properties of digitalis-like compounds predicted by ChemBioOfï¬ce software; Figure S1: Visualization of whole transcriptome data of BC-PAP treated with 50 µM proscillaridin A for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S2: Visualization of whole transcriptome data of FTC133 treated with 50 µM digoxin for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S3: Visualization of whole transcriptome data of FTC133 treated with 50 µM digoxigenin for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S4: Visualization of whole transcriptome data of FTC133 treated with 50 µM proscillaridin A for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S5: Visualization of whole transcriptome data of FTC133 treated with 50 µM strophantin K for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S6: Visualization of whole transcriptome data of TPC-1 treated with lanatoside C for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S7: STRING protein interaction networks of significantly up- or downregulated genes by digitalis-like compounds in all three cell lines and at all three time points; Figure S8: Bonferroni corrected P-value enrichment scores of significantly upregulated pathways by digitalis-like compounds combined for all three cell lines and for all three time points. P-values are generated by using the Gene Ontology (GO) platform category Biological Processes; Figure S9: Expression of JUN after treatment for 48 and 72 hours of BC-PAP, FTC133 and TPC-1 with the indicated digitalis-like compounds. Data are obtained from three independent experiments. Data are means {plus minus} SD. Square boxes represent statistical output of Spearman's rho tests for degree of correlation between expression of JUN with hNIS expression at 48 and 72 hours time points; Figure S10: Gene expression of thyroid transcription factors TTF1, TTF2 and PAX8 in BC-PAP, FTC133 or TPC-1 after treatment with the indicated digitalis-like compounds for 48 or 72 hours. Data are obtained from three independent experiments. Data are means {plus minus} SD; Figure S11: Heatmaps depicting gene expression of ABC transporters and organic anion transporters in untreated and digitalis-like compound treated BC-PAP, FTC133 and TPC-1 (N=6); Figure S12: Full unedited Western blot pictures, cropped pictures are depicted in Figure 1B.

PDF file - 55K, Consort flow chart.

Video made with the laparoscopic fluorescence camera during experiment #1.

Supplementary Table 2 PDF file - 30K, Patient characteristics and historical controls

Biodistribution of Iodine-131-girentuximab-IRDye800CW and the irrelevant control Iodine-125-IgG-IRDye800CW in tumor and normal tissue samples. Left: first control experiment (#6). The mean tumor-to-normal-ratio for girentuximab and the irrelevant control antibody were 6 and 1, respectively. Right: second control experiment (#7). The mean tumor-to-normal-ratio for girentuximab and the irrelevant control antibody were 10 and 1, respectively.

Supplementary Figure 1 PDF file - 34K, Supplementary Figure 1 CliniMACS-based pDC isolation is feasible

Supplementary Materials- figure legends

In patients with colorectal peritoneal metastases scheduled for cytoreductive surgery, accurate preoperative estimation of tumor burden and subsequent intraoperative detection of all tumor deposits remains challenging. In this study (ClinicalTrials.gov NCT03699332) we describe the results of a phase I clinical trial evaluating [ 111 In]In-DOTA-labetuzumab-IRDye800CW, a dual-labeled anti-carcinoembryonic antigen (anti-CEA) antibody conjugate that enables both preoperative imaging and intraoperative radioguidance and fluorescence imaging. Primary study outcomes are safety and feasibility of this multimodal imaging approach. Secondary outcomes are determination of the optimal dose, correlation between tracer uptake and histopathology and effects on clinical strategy. Administration of [ 111 In]In-DOTA-labetuzumab-IRDye800CW is well-tolerated and enables sensitive pre- and intraoperative imaging in patients who receive 10 or 50 mg of the tracer. Preoperative imaging revealed previously undetected lymph node metastases in one patient, and intraoperative fluorescence imaging revealed four previously undetected metastases in two patients. Alteration of clinical strategy based on multimodal imaging occurred in three patients. Thus, multimodal image-guided surgery after administration of this dual-labeled tracer is a promising approach that may aid in decision making before and during cytoreductive surgical procedures.

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.

Sunitinib is an effective treatment for patients with metastatic Renal Cell Carcinoma (mRCC) but ultimately resistance occurs. The aim of this study was to investigate sunitinib resistance in RCCs and to develop therapeutic combination strategies with targeted radioimmunotherapy (RIT). We studied two RCC models, analyzed Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) and AXL/MET expression and performed therapy studies in Balb/cnu/nu mice combining sunitinib and [177Lu]Lu-cG250 RIT (6.5 MBq/10 μg), specifically targeting RCC cells. pAXL and pMET were expressed in sunitinib-resistant SK-RC-52 and absent in sunitinib-sensitive NU12. NGS evaluation showed that expression of VEGFA, VEGFB, VEGFD, PGF and VEGFR1,2,3 was higher and expression of VEGFC and PDGFA was lower in NU12 than in SK-RC-52. Therapy studies combining sunitinib with [177Lu]Lu-cG250 RIT showed that the best response in mice with "resistant" SK-RC-52 tumors was observed with two cycles of Sunitinib and [177Lu]Lu-cG250 RIT, probably due to increased vascular permeability by sunitinib treatment. In the "sensitive" NU12 model, two cycles of [177Lu]Lu-cG250 RIT and two cycles of combination treatment were equally effective. Enhanced therapeutic efficacy was achieved when two agents ([177Lu]Lu-cG250 RIT and sunitinib) that on their own did not induce satisfactory response levels, are combined. Our findings provide a promising new therapeutic strategy for patients with advanced RCC.

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rβγ > IL-2 Rβγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rβγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.