We initially investigated the sensitivity of seven different ABC-DLBCL cell lines (HBL1, OciLy-3, Oci-Ly10, RIVA [RI-1], SU-DHL-2, TMD-8, U-2932) to treatment with ¹⁷⁷Lu-lilotomab satetraxetan. Cells were treated for 18 h with 11 different doses of ¹⁷⁷Lu-lilotomab satetraxetan ranging from concentrations of 0.01–20 μg/ml (specific activity: 600 MBq/mg), washed and plated in 96-well plates. Mock treated cells were included as controls. The total DNA content in each well was assessed using the CyQuant reagent as a readout of cell proliferation. Comparative analysis of relative proliferation capacity compared to untreated control cells identified U-2932 and the RIVA cells as the most resistant cell lines, showing over 40% of signal intensity of untreated control cells even after treatment at 20 μg/ml ¹⁷⁷Lu-lilotomab satetraxetan (Figure 1A). Conversely, Oci-Ly10 cells showed highest sensitivity to treatment with a 70% decreased proliferation capacity in response to treatment with 0.25 μg/ml ¹⁷⁷Lu-lilotomab satetraxetan. Notably, CD37 mRNA and CD37 surface expression were not associated with the resistance to CD37-target RIT (Table 1). We confirmed the differential sensitivity of these three cell lines in a metabolic cell viability assay, utilizing MT RealTimeGlo, that allowed the monitoring of cell proliferation throughout a continuous period of 72 h (Figures 1B,C). Cells were treated as previously and the luminescent assay substrate added 72 h after plating into micro-well titer plates. All cell lines and control treatment groups showed continuous proliferation throughout the observation period. Addition of cold, non-¹⁷⁷Lu chelated lilotomab (HH1-DOTA) did not markedly inhibit proliferation in either cell line. Oci-Ly10 cells were sensitive to even the lowest tested dose of 0.05 μg/ml ¹⁷⁷Lu-lilotomab satetraxetan and ceased proliferation at 0.25 μg/ml. Confirming the observed resistance in the CyQuant assay, U-2932 and RIVA retained ~60 and 40%, respectively, of the proliferation capacity of untreated cells at 5 days after treatment with 2 μg/ml ¹⁷⁷Lu-lilotomab satetraxetan. Again, RIVA cells were more sensitive to ¹⁷⁷Lu-lilotomab satetraxetan than U-2932 and showed about 60% of the proliferation capacity of control cells at a dose of 0.5 μg/ml, which is half of the dose required in U-2932 cells to reach a similar level of inhibition.

To conclude, U-2932 and RIVA have been shown to be ¹⁷⁷Lu-lilotomab satetraxetan treatment resistant ABC-DLBCL cell lines. Furthermore, the resistance of these cell lines to CD37-targeted RIT was not due to a reduced expression of and binding to CD37 on the cell surface.

Only two out of seven cell lines of the ABC-DLBCL panel did not respond well to CD37-targeted RIT. These two cell lines, U-2932 and RIVA, may represent models of most challenging to treat DLBCL. To understand and overcome the lack of response to ¹⁷⁷Lu-lilotomab satetraxetan treatment, we set out to find combinatorial drug partners for ¹⁷⁷Lu-lilotomab satetraxetan capable of reversing CD37-targeted RIT resistance in both cell lines. U-2932 and RIVA cells were treated with 1 and 0.5 μg/ml of ¹⁷⁷Lu-lilotomab satetraxetan for 18 h, respectively, washed and seeded onto micro-well plates pre-printed with a library of 384 anti-cancer compounds (Cambridge anti-Cancer Compound library; SelleckChem). Three days post-plating RealTimeGlo reagent was added and luminescence read on three consecutive days to assess the relative amount of metabolically active cells. The screen design is schematically presented in Figure 2.

Both cell lines continuously proliferated throughout the observation period, albeit U-2932 cells showed a higher growth rate and stronger resistance to ¹⁷⁷Lu-lilotomab satetraxetan monotreatment than RIVA cells in the primary screen (Supplementary Figure 1). Three different concentrations of drugs (U-2932: 10/1,000 nM; RIVA: 10/100 nM final concentration) were tested to account for compound-specific differences in potency. Inhibitory compounds were considered as a hit candidate if they: (1) in combination with ¹⁷⁷Lu-lilotomab satetraxetan inhibited cell proliferation over two consecutive days to a degree greater than the expected additive effect of the mono-treatments alone (Bliss theorem, see Materials and Methods for details), and (2) the inhibitory effect of the compound alone at the tested concentration was <90% relative to the untreated control. These criteria were met by 53 compounds in U-2932 (11 at 10 nM and 42 at 1 μM) and 27 compounds in RIVA cell lines (8 at 10 nM and 19 at 100 nM) (Tables 2, 3). Figures 3A,B summarize the screen results for each cell line and library concentration. Hit candidates are highlighted in dot-plots showing the relative proliferation of cells at day 5 treated with the compound alone vs. its combination with ¹⁷⁷Lu-lilotomab satetraxetan (datasets are included as excel spreadsheets in Supplementary Material). Topoisomerase inhibitors accounted for 13% of the hits in U-2932 and 23% in RIVA, and histone deacetylase (HDAC) inhibitors for 7% of the hits in U-2932 and 27% in RIVA cells (Tables 2, 3). The enrichment defined topoisomerases and histone deacetylases (HDAC) as prime targets for co-inhibition in both cell lines. A third prominent group of hit candidates comprised inhibitors targeting mitotic cell cycle kinases, including AURKA, AURKB, CDK1, PLK1, and WEE1 (17% of total hits in U-2932; 12% in RIVA).

Since both topoisomerase inhibitors, such as doxorubicin or etoposide, and HDAC inhibitors are known to cause direct or indirect DNA damage, respectively, it is likely they might overcome resistance by potentiating the level of DNA damage in ¹⁷⁷Lu-RIT-targeted cells (24–26). Therefore, we focused on the third group of compounds, the mitotic kinase inhibitors, that affect kinases with critical functions for both mitotic entry and exit, and have a role in termination of the DNA damage-induced G₂-checkpoint (27–29). In particular we further explored the combination of ¹⁷⁷Lu-lilotomab satetraxetan with the dual CDK1/2-Aurora A/B inhibitor JNJ-7706621, the Aurora A inhibitor alisertib (MLN8237), and the Plk1 inhibitor GSK461364, respectively. JNJ-7706621 and alisertib were hit candidates in both cell lines. GSK461364 did not score as a hit candidate in U-2932 cells only due to its high potency as a mono-therapy.

To investigate the validity of the synergism observed following ¹⁷⁷Lu-Lilotomab satetraxetan with the effect of the cell cycle kinase inhibitors in resistant cell lines, U-2932 cells were treated or not with ¹⁷⁷Lu-lilotomab satetraxetan (0.5, 1, and 2 μg/ml) for 18 h, washed and seeded onto 384-well plates pre-printed with 11-step gradients of JNJ-7706621, alisertib, and GSK461364 ranging from 0 to 1,280 nM. Similar to the primary screen, viability was measured by RealTimeGlo. Dose-response profiles were recorded at day 5 and Combination Indexes (CI) calculated to test for a synergistic interaction of ¹⁷⁷Lu-Lilotomab satetraxetan-inhibitor combinations (Chou-Thalaly; CompuSyn software). CI values were calculated for combinations within the minimum and maximum effect range of mono-treatment of each inhibitor.

Figure 4 shows dose-response curves for treatment with either drug alone or in combination with different doses of ¹⁷⁷Lu-lilotomab satetraxetan. The anti-proliferative effect of the dual CDK1/2-AURKA/B inhibitor JNJ-7706621 was moderate, even at high doses (Figure 4A). However, and confirming the primary screen results, the combination with ¹⁷⁷Lu-lilotomab satetraxetan led to a greater reduction in the fraction of viable cells than each treatment alone. Synergism (CI <1) was observed for all tested combinations (blue circles in Fa/CI plots). The AURKA inhibitor alisertib had a bi-phasic dose-response profile, with a decreasing anti-proliferative effect at doses above 160 nM (Figure 4B) and synergism was observed within a range of 10–160 nM. U-2932 cells were highly sensitive to treatment with the PLK1 inhibitor GSK461364 with a near complete growth inhibition obtained at 20 nM (Figure 4C). At lower doses the sensitivity was greatly reduced, and when combined with ¹⁷⁷Lu-lilotomab satetraxetan growth inhibition was, however modestly, potentiated. Weak synergism (CI 0.75–0.95) was observed only at GSK461364 concentrations near the maximum effect (20 and 40 nM; Fa close to 1). Similar CI index profiles were obtained in RIVA cells (Supplementary Figure 2). The results of the validation screen identified the dual CDK1/2 and AURKA/B kinase inhibitor JNJ-7706621 as the best hit candidate. We hence controlled first for target-specificity/activity for inhibition of CDK1 and AURKB (Supplementary Figure 3). Synergism with ¹⁷⁷Lu-lilotomab satetraxetan was then confirmed in two additional combination experiments in U-2932 cells covering the range of 100–10,000 nM JNJ-7706621 (Figure 4A, yellow and red circles).

The validation experiments confirmed a synergistic drug interaction of JNJ-7706621 and ¹⁷⁷Lu-lilotomab satetraxetan in the inhibition of proliferation of U-2932 and RIVA cells, as assessed by the decreased capacity in reducing the RealTimeGlo substrate. CD37-targeted RIT induced DNA damage and inhibition of CDK1/2 and AURKA/B kinases by JNJ-7706621 are independently of each other expected to result in cell cycle progression defects. For instance, in mitotic shake-off synchronized HeLa cells, inhibition of CDK1/2 and AURKA/B by JNJ-7706621 at a final concentration of 1–3 μM was reported to delay exit from G₁, to arrest cells at G₂/M, and to induce endoreduplication (30). We thus investigated the effects of mono- and combination therapy on cell cycle progression in U-2932 and RIVA cells (Figure 5). Cells were treated with or without ¹⁷⁷Lu-lilotomab satetraxetan (18 h; U-2932: 1 μg/ml, RIVA 0.5 μg/ml), washed and treated with or without 500 nM JNJ-7706621, a concentration at which strong synergism was observed in both cell lines (Figure 4 and Supplementary Figure 2).

Samples were taken immediately after treatment with ¹⁷⁷Lu-lilotomab satetraxetan and at 24, 72, and 144 h after inhibitor addition, and DNA content and cell death assessed by flow cytometry (Figure 5A). Medium was replenished (with or without inhibitor) at 72 h to allow for optimal growth conditions throughout the observation period. Treatment with JNJ-7706621 alone induced a prominent but transient accumulation of cells in G₂/M-phase (4n DNA content) after 24 h of treatment in both cell lines (Figure 5B). Similarly, pre-treatment with ¹⁷⁷Lu lilotomab satetraxetan alone also induced a prominent but transient accumulation of cells in G₂/M-phase in both cell lines. The addition of JNJ-7706621 to ¹⁷⁷Lu-lilotomab satetraxetan treated cells strongly reduced the fraction of cells in G₁ phase (2n) at the 24 h time time-point and at later times strongly increased appearance of single cells with <2n or >4n DNA content, indicative of cell death and endoreduplication/cytokinesis failure, respectively. We therefore quantitatively assessed the fraction of endoreduplication (>4n DNA content), cell death (Pacific blue uptake), as well as cell size (median forward scatter) in both cell lines in two independent experiments (Figure 6 and Supplementary Figures 4, 5). Combination treatment strongly induced the formation of endoreduplicating cells (20- and 12-fold increase in U-2932 and RIVA, respectively; Bliss CI U-2932 = 0.55, RIVA = 0.69; [CI = ((E_RIT + E_JNJ)–E_RIT × E_JNJ)/E_[RIT+JNJ]]), which was accompanied by a 2-fold increase in relative cell size, compared to untreated and mono-treated cells. Visual inspection of cells by microscopy confirmed this result, revealing prominent occurrence of multinucleated cells and increased DNA content (Supplementary Figure 6). Most importantly, combination treatment led to a 5- and 13-fold increase in cell death compared to untreated cells at the 144 h time point in U-2932 (CI = 0.72) and RIVA (CI = 0.50), respectively. To further explore the mechanistic details behind the anti-tumor activity of JNJ-7706621 and ¹⁷⁷Lu-litomab satetraxetan combination treatment, we used the same experimental setup as described in Figure 5 but now monitored PARP cleavage, a late apoptotic event, and cell growth (Figure 7). In accordance with our RealTime-Glo data we found that cells exposed to the combination showed a significantly reduced growth rate between 24 and 72 h post-treatment as compared to control cells and cells receiving monotherapy (Figure 7A). The fraction of cells in late apoptosis (cleaved PARP positive cells), was at all tested time-points significantly increased in cells receiving combination therapy as compared to control cells, reaching about 50% after 144 h (Figure 7B). Induction of apoptosis was also significantly increased in cells receiving single agent therapy at 72 and 144 h time points compared to control. At 144 h, 22% of JNJ-7706621 and 28% ¹⁷⁷Lu-litomab satetraxetan treated cells were determined as late apoptotic, indicating an additive effect of the combination treatment. Taken together, these results suggest that the synergistic anti-proliferative effect of the combination of JNJ-7706621 and ¹⁷⁷Lu-litomab satetraxetan is a consequence of JNJ-7706621 mediated mitotic infidelity of cells which have over-come the ¹⁷⁷Lu-litomab satetraxetan induced G₂-arrest, leading to incompatibility with proliferation and to excessive cell death by apoptosis (Figure 7C).

ABC-DLBCL cell lines were maintained in RPMI 1640-GlutaMAX medium (Gibco 61870-044) supplemented with 15% fetal bovine serum (Biowest S181B-050) and 1% penicillin-streptomycin (Gibco 15140-122) at 37°C in a humidified atmosphere containing 5% CO₂. Cell lines were obtained as previously described (21) and their identity was authenticated by short tandem repeat DNA profiling (IDEXX BioResearch, Ludwigsburg, Germany). Real Time Glo was from Promega (G9713). The Selleck Cambridge Cancer Compound library was obtained from and printed onto 384-well plates [384 well, PS, F-bottom, μclear, white, lid, sterile, Greiner Bio-One 781098 (82050-076)] by the High-Throughput Chemical Biology Screening Platform at the Center for Molecular Medicine Norway (NCMM). Antibodies used were: rabbit-anti-phospho-BRCA2(Ser3291) (AB9986 Millipore), rabbit-anti-Cleaved-PARP (Asp214) Alexa647-conjugate (#6987, Cell Signaling), rabbit-anti-phospho-histone 3-Ser10 (06-570, Millipore), Alexa488 anti-rabbit (A21206, Life Technologies), and Alexa647 donkey-anti-mouse (Jackson ImmunoResearch, UK). The DNA stain FxCycle™ Far Red (200 nM FxCycle and 0.1mg/ml RNase A) (Thermo Fisher Scientific) was used together with Pacific Blue (Thermo Fisher Scientific, P10163) staining, and Hoechst 33258 (Sigma-Aldrich) (1.5 μg/ml) in other experiments.

The chelator (p-SCN-Bn-DOTA, Macrocyclics, TX, USA) was dissolved in 0.005 M HCl, added to the HH1-DOTA (lilotomab) in a 6:1 ratio and pH-adjusted to ~8.5 using carbonate buffer. After 45 min of incubation at 37°C, the reaction was stopped by the addition of 50 μL per mg of Ab of 0.2 mol/L glycine solution. To remove free p-SCN-Bn-DOTA, the conjugated antibody was washed using Vivaspin 20 centrifuge tubes (Sartorius Stedim Biotech, Göttingen Germany) 4–5 times with NaCl 0.9%. Before labeling with ¹⁷⁷Lu, the pH was adjusted to 5.3 ± 0.3 using 0.25 mol/L ammonium acetate buffer. Between 120 and 220 MBq of ¹⁷⁷Lu (ITG, Garching, Germany) was added to 1 mg of satetraxetan-Ab and incubated for 15–30 min at 37°C. The radiochemical purity (RCP) of the conjugate was evaluated using instant thin-layer chromatography. If RCP was below 95% the conjugate was purified by elution through a Sephadex G-25 PD-10 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

Cells were treated in 6-well plates (2.5 × 10⁶ cells/mL) without shaking for 18 h with Betalutin (specific activity of ~600 Mbq/mg) at a final concentration of 1 μg/ml for U2932 and 0.5 μg/ml for RIVA (or as otherwise specified). After treatment, PBS was added to the cells, and the cells pelleted. Cells were first resuspended in 1 ml PBS, then washed twice in PBS and finally diluted in growth medium to desired concentration for the assay applied.

For the initial screening of ABC-DLBCL cell lines cells were incubated in deep well plates (Nunc™ 96-Well Polypropylene DeepWell Storage Plates from Thermo Scientific) with 12 different concentrations of ¹⁷⁷Lu-lilotomab satetraxetan, including a control with no treatment, for 18–20 h with shaking. Cells were washed three times with PBS using a plate washer (ELx405 Select Deep Well Washer from BioTek) and seeded in 96-well plates in 0.2 ml medium and incubated at 37°C/5% CO₂. Fifty microliter of fresh medium was added after 72 h. The cytotoxic effect was measured at 144 h using the CyQuant NF Cell Proliferation Assay kit (ThermoFischer) for Ascent FL multiplate reader (ThermoFischer. IC50 were calculated using Prism (GraphPad) and clustering analysis performed in J-Express Pro (49).

Cells treated with ¹⁷⁷Lu-lilotomab satetraxetan and untreated control cells were seeded onto 384-well plates pre-printed with the 384-compound Cambridge Cancer Compound library sourced from SelleckChem. The library was divided onto two plates and a total of 48 no-drug controls were included per plate. Cell seeding densities were 120 000 cells/mL, and a volume of 25 μL was seeded in each well-giving 3,000 cells/well. The drug library was screened at two concentrations for each cell line (10/100 nM for RIVA and 10/1,000 nM for U-2932). Three days after seeding, 25 μL of diluted NanoLuc® luciferase and MT Cell Viability Substrate (RealTime-Glo) was added to each well. Cells were incubated with the reaction mix for 1 h at 37°C before measuring luminescence in a Tecan Spark multimode microplate reader, with integration time set to 1 s. Luminescence readings were repeated each day for three consecutive days. A microplate sample processor (Precision XS, BioTek) was used to facilitate the cell seeding and dispensing of RealTimeGlo reagent. Hit candidates were identified using the Bliss Independence test for synergy. The effect of each drug alone (Fa) at each concentration was calculated as the fraction of dead cells as compared to control cells

a similar calculation was performed for the average effect of ¹⁷⁷Lu-lilotomab satetraxetan alone (Fb¯)

Through the following equations we found the expected additive effect (E) and the measured effect (M) of the combination of drug + ¹⁷⁷Lu-lilotomab satetraxetan:

The Bliss score was defined as:

The normalization to the fraction of viable cells in samples treated with drug alone (1-Fa in equation above) was carried out to compensate for the reduced population of cells that could be affected by combined treatment with ¹⁷⁷Lu-lilotomab satetraxetan. The standard deviation for the effect of ¹⁷⁷Lu-lilotomab satetraxetan alone in the 48 control wells was calculated on each plate as follows:

Drugs with a Bliss score two times higher than the standard deviation of ¹⁷⁷Lu-lilotomab satetraxetan treated controls were scored as potential hits. Finally, we excluded inhibitory drugs that alone reduced viability by >90% to that of untreated control. Each plate was treated individually. In validation experiments the same procedure was used with the following modifications; Cells were treated in 12-well plates (1.2 ml per well, with 2.5 × 10⁶ cells/ml) without shaking for 18 h with ¹⁷⁷Lu-lilotomab satetraxetan at final concentrations of 0.5, 1, and 2 μg/ml for U2932 and 0.25, 0.5, and 1 μg/ml for RIVA. After treatment, cells were seeded on 384-well plates pre-printed with JNJ-7706621, Alisertib and GSK461364 in an 11 step concentration gradient (0-1280 nM). A total of 60 no-drug controls were included on each plate. Synergy of drug/¹⁷⁷Lu-lilotomab satetraxetan combinations was determined by calculating Combination Indexes (CI) using the Chou-Talaly theorem (CompuSyn software) (50), where CI < 1 represents synergy. In replicates of validation experiment cells were treated with ¹⁷⁷Lu-lilotomab satetraxetan in 96 deep-well plates (100 μL per well, with 2.5 × 10⁶ cells/ml) for 18 h, diluted to 40 cells/μL and 25 μL transferred to 384-well plates. Immediately after seeding a Tecan D300 Digital dispenser was used to administer drug to wells at 100, 266, 707, 1,880, and 5,000 nM (f.c. experiment 2) or 200, 532, 1,410, 3,760, and 10,000 μM (f.c experiment 3).

For live/dead discrimination, ¹⁷⁷Lu-lilotomab satetraxetan treated cells were diluted to 500 000 cells/mL, transferred to T-25 cell culture flasks and inhibitor (JNJ-7706621) added to a final concentration of 500 nM. ¹⁷⁷Lu-lilotomab satetraxetan treated and untreated control samples, with or without inhibitor, were harvested for flow cytometry analysis before and at 24, 72, and 144 h after addition of the inhibitor (Figure 5A). At 72 h, 6 mL of fresh medium w/wo inhibitor was added to allow continuous growth of the cells that were harvested at the latest timepoint (144 h). Before fixation, cell pellets were resuspended in 200 μL PBS with 18 ng/μL of Pacific Blue and incubated at 4°C for 20 min for live/dead discrimination. One milliliter of PBS was added to the samples, and cells were pelleted and resuspended in 1 mL of ice-cold 70% ethanol for fixation. Samples were stained with FxCycle Far Red for visualization of DNA, and analyzed on an LSR II flow cytometer (BD Biosciences) using FlowJo software. The same experimental setup (omitting Pacific Blue) was followed for assessment of PARP cleavage. Cells were fixed in 1.5% neutral buffered formaldehyde for 5 min at r.t, washed in PBS, and stored in Methanol (−20°C). For analysis, cells were rehydrated and stained with anti-cleaved-PARP-Asp214 and Hoechst. Cell counts in Figure 7A were determined in quadruplicates for each time-point using a Countess Automated Cell counter (Thermo Fisher). For testing efficacy of JNJ-7706621, cells were arrested in mitosis by culturing for 16 h in medium containing nocodazol (0.04 μg/ml). Still in the presence of nocodazol, cells were treated with JNJ-7706621 for 1 or 6 h at a final concentration of 250, 500, or 1,000 nM before fixation in ice-cold 70% ethanol. Samples were stained with primary antibodies against CDK and Aurora B targets phospho-BRCA2-Ser3291 and phospho-histone H3-Ser10, respectively.

CD37-expression in U-2932, RIVA and Oci-Ly10 was measured by determination of the binding capacity of cold antibody (HH1-dota). For accurate comparison of different cell lines, we included barcoding with CellTracer stain and pooled the samples from the different cell lines together in a single tube before staining with the CD37 antibody. To this, 2 × 10^e6 cells were pelleted and resuspended in 1 ml PBS and labeled with CellTrace stain (U-2932: CFSE 1 μM f.c., RIVA: Violet 2 μM f.c., Oci-Ly10: blank) in the dark for 20 min at 37°C with gentle shaking every 3–5 min. Cells were diluted in 5 ml of pre-warmed medium. After 5 min, cells were pelleted and resuspended in 1 ml fresh medium, pooled and an additional 1 ml of medium added. Cells were kept on ice thereafter. The cell mixture was divided into four polystyrene tubes and pelleted. Primary antibody (HH1-dota) was diluted in medium and added to three of the four tubes (no antibody, 1, 2, and 20 μg). Cells were incubated on ice for 20 min, washed twice with cold PBS and resuspended in medium containing Alexa647 donkey-anti-mouse (Jackson ImmunoResearch, UK) at 1 μl/ml. After 20 min incubation on ice, cells were resuspended in 1 ml cold PBS w/1% Fetal Bovine Serum and analyzed on a LSR II flow cytometer.

Cells were imaged using a CellObserver microscope system (Carl Zeiss) equipped with a 20×/0.8 PlanApo Phase 2 lens, a Hamamatsu ORCA-Flash4.0 v3 camera, a temperature controlled XL-chamber, a temperature, humidity and CO₂ controlled stage incubator, a motorized coded X,Y-stage, a Definite Focus system and a HXP120 Metal-Halide illumination unit. Visual inspection of cells prepared for flow cytometry was conducted using 96-well flat-bottom plates (Greiner Bio-One, Kremsmünster, AUT).

SP: Nordic Nanovector ASA: Employment, Patent. KM, RG, and JD: Nordic Nanovector ASA: Employment, Equity Ownership, Patents. GR: Patent. RS: Institutional research funds from Nordic Nanovector ASA, Patent. FB: Institutional research funds from Acerta, ADC Therapeutics, Bayer AG, Cellestia, CTI Life Sciences, EMD Serono, Helsinn, ImmunoGen, Menarini Ricerche, NEOMED Therapeutics 1, Nordic Nanovector ASA, Oncology Therapeutic Development, PIQUR Therapeutics AG; consultancy fee from Helsinn, Menarini; expert statements provided to HTG; travel grants from Amgen, Astra Zeneca, Jazz Pharmaceuticals, PIQUR Therapeutics AG. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.