Approximately 50% of children and adolescents diagnosed with soft tissue sarcoma suffer from rhabdomyosarcoma (RMS), a striated-muscle lineage malignancy with variable pathologies [1
]. The two major subtypes of the tumor are embryonal RMS (ERMS) and alveolar RMS (ARMS), accounting for 60% and 20% of all cases, respectively. ARMS is more aggressive and is characterized by a chromosomal translocation resulting in a PAX3-FOXO1
gene fusion, whereas ERMS is associated with different tumor-promoting mutations and chromosome number aberrations [2
]. Surgery, radiation, and multi-drug chemotherapy composed of vincristine (VCR), actinomycin D and cyclophosphamide are the standard treatments for RMS [1
]. The overall survival rates for RMS patients have improved within the last few decades but the prognostic outcome is still very poor for high-risk patients, including those presenting metastatic diseases, ARMS subtype, or diagnosis in adulthood [3
]. The treatment goes along with high toxicity and many who survive RMS will experience long-term adverse effects as adults [5
]. Therefore, there is an urgent need for new targeted therapies to improve overall survival rates, and to overcome long-term side effects.
Nanovesicle-mediated chemotherapeutic drug delivery offers the possibility to increase the therapeutic effect in the tumor and to decrease side effects in healthy tissues [6
]. Passive accumulation of nanoparticles in the tumors has been attributed to the so-called “enhanced permeability and retention” (EPR) effect [10
]. Fast-growing solid tumors display a leaky vascular architecture and a lack of functional lymphatics, enabling the size-dependent passive extravasation and accumulation of nanoparticles in the interstitial space of the tumor [11
]. Most recently, this dogma has been challenged by findings showing that the great majority of nanoparticles enter tumors using an active process through endothelial cells [12
]. Liposomal formulations of chemotherapeutic drugs have demonstrated the safety and improved pharmacokinetic properties of the drug [13
]. Prominent examples are liposomal doxorubicin (Doxil), daunorubicin (DaunoXome), and VCR (Marqibo) which have contributed to reducing side-effects compared to the free drug [14
]. However, liposomal formulations have not, so far, been able to increase the therapeutic effect of the encapsulated drug. One possibility to achieve this is to modify the liposomal surface with tumor targeting-ligands, such as peptides [17
], antibodies or antibody fragments [18
], for active targeting to cancer cells. Single-domain antibodies (sdAb), first discovered in camelids [19
], are the smallest possible antibody fragments (15 kDa) derived from heavy-chain antibodies. They are characterized by affinities comparable to conventional bivalent antibodies, as well as by high solubility, tissue penetration, and stability [20
]. Previously, we developed the optimal formulation of liposomal VCR [21
], and we investigated its pharmacokinetic and biodistribution in a mouse model engrafted with human RMS cells, revealing longer plasma circulation time and enhanced tumor accumulation of the liposomal drug compared to free VCR. Now, to further improve tumor accumulation of the liposomes in RMS by active targeting, we selected and investigated novel RMS-targeting sdAb.
The fibroblast growth factor receptor 4 (FGFR4) belongs to the family of receptor tyrosine kinases and is involved in myogenesis and muscle regeneration by promoting cell survival and differentiation [22
]. FGFR4 is absent in normal differentiated muscles and is specifically overexpressed in RMS [24
], as well as in other tumors, such as hepatocellular carcinomas, head and neck squamous cell carcinomas and basal-like breast cancer [25
]. Therefore, FGFR4 represents a promising candidate for targeted therapies in RMS.
Another approach that could benefit from specific tumor targeting and that may improve the therapeutic outcome for RMS patients, is represented by chimeric antigen receptor (CAR) T cells. These cytolytic T cells are engineered with an extracellular antigen-binding domain recognizing specifically surface antigens on tumor cells. The intracellular part of the receptors is composed of T cell receptor signaling and costimulatory domains [29
]. Tremendous clinical success has been achieved in the treatment of hematological malignancies with CAR T cells targeting CD19 [30
], CD22 [32
] and the B cell maturation antigen (BCMA) [33
]. The application of CAR T cells for solid tumors has been more challenging, due to the lack of ideal tumor-specific target molecules, and also due to the strong immunosuppressive tumor microenvironment (TME) of solid tumors. Nevertheless, preclinical studies of CD276 (B7-H3) CAR T cells in pediatric solid tumors demonstrated good activity [34
], and encouraging results have been reported for RMS CAR T cells targeting HER2 led to remission in a child with refractory metastatic RMS [35
Here, we selected FGFR4-binding sdAb from two fully synthetic phage display libraries [36
]. After validation, sdAb were coupled to the surface of VCR-loaded liposomes and tested as a potential drug-delivering platform for RMS cells in vitro. Moreover, we generated FGFR4 targeting CAR T cells with the selected sdAb and examined their cytotoxic potential for RMS cells in vitro.
In this study, we developed three therapeutic strategies for RMS by targeting FGFR4 with sdAb and validated them on RMS cells. We selected four FGFR4 binding sdAb and tested them in vitro for (a) inhibitory activity of FGFR4 signaling; (b) active drug delivery as liposome conjugates, and (c) cell-mediated immunotherapy as CAR constructs.
The four selected sdAb A8, B1, B5 and F8 not only bind to FGFR4 expressed on RMS cells but are also able to block the FGF19-FGFR4-MAPK signaling axis. In ARMS, FGFR4 is a direct target gene of the fusion protein PAX3-FOXO1 [41
], and in ERMS FGFR4 is frequently amplified with 12% of the tumors harboring activating mutations of the receptor [42
]. In RMS, besides overexpression, FGFR4 has been shown to harbor activating mutations in over 6% of all tumors, resulting in constitutive tumor-promoting signaling within the cells [2
]. Although we did not observe a toxic effect on cultured RMS cells, it is tempting to speculate that FGFR4 signaling could still represent a therapeutic target for sdAb in RMS. Moreover, FGFR4 is not only implicated in RMS tumorigenesis, but drives tumor progression in FGF19 expressing hepatocellular carcinomas, head and neck squamous cell carcinomas, and basal-like breast cancer [25
]. It is also estimated that 0.5% of all tumors display abnormalities in FGFR4 [47
]. The selected sdAb could therefore also serve as possible therapeutic approach for cancers other than RMS.
Surface plasmon resonance spectroscopy of sdAb binding to FGFR4 revealed strong affinities in the order of nano- to picomolar. The measured data could not be fitted with a 1:1 binding model. Best fits were obtained with the heterogeneous ligand model indicating two separate binding affinity parameters for the sdAb to FGFR4. We had to directly immobilize recombinant FGFR4 to activated carboxyl groups on the sensor chip through amine group binding. Binding of FGFR4 through a His-tag or biotin–streptavidin linker was not compatible with our measurements, since analytes and ligands contained a 6xHis-tag, and FGFR4 biotinylation led to interference with sdAb binding. Therefore, it is possible that the non-oriented binding of FGFR4 to the sensor chip could have led to a partial or complete steric hindrance of the sdAb binding site, resulting in heterogeneous binding parameters. This is obvious when comparing Rmax values, representing the maximal sdAb binding signal: for the affinity measurements of A8, we could immobilize 800 RU FGFR4 to the sensor chip. With approximate molecular weights of 40 kDa for the ligand and 17 kDa for the sdAb, we would expect an Rmax of 340 RU ((MWFGFR4/MWNB) × 800 RU) but ultimately a value of only 44 RU was achieved. Since we were not able to fully regenerate the flow cells after sdAb binding, we performed all measurements with freshly immobilized FGFR4 for each sdAb. This resulted in different amounts of immobilized FGFR4. A8, B1 and B5 analysis was performed with approximately 800 RU of FGFR4 whereas for F8 and mCh we immobilized 9000 and 12,000 RU, respectively. The measurement of negative control mCh on such high ligand densities forced unspecific interactions at high sdAb concentrations, and this resulted in low calculated affinities compared to the selected FGFR4 sdAb.
Both free and liposome-conjugated sdAb bound specifically to Rh4-FR4wt cells and showed, except for uncoupled B5 sdAb, no binding to Rh4-FR4ko. Nevertheless, recombinant B5 binding to Rh4-FR4ko cells in FACS experiments was only 0.25 times higher than mCh control sdAb, whereas on Rh4-FR4wt it was 2 times higher. Affinity measurements revealed binding of only A8 and B5 another FGFR-member, FGFR2. The binding affinity of A8 to FGFR2 was in the micromolar range and therefore very low when compared to the binding affinity to FGFR4. The fast koff rates further highlighted its weak binding to FGFR2. In contrast, B5 showed high affinities to FGFR2 in the low nanomolar range, thus similar to its affinity for FGFR4. Rh4 cells do express FGFR2, but protein levels are lower compared to FGFR4. Moreover, the Rh4-FR4ko cells have reduced FGFR1 and FGFR2 protein levels compared to Rh4-FR4wt. The reasons for the lower expression level of FGFR1 and FGFR2 in Rh4-FR4ko are not completely clear but could be due to a clonal effect or to regulatory loops. Therefore, it is well possible that the binding of sdAb A8 and B5 to cell surface FGFR2 would be only detectable above a certain expression level.
The formulation of liposomal VCR was modified from the previously established one [21
] by the introduction of DSPE-PEG-maleimide at 1 mol%. As expected, the resulting physico-chemical properties of the liposomes and the drug loading efficiency were comparable. SdAb coupling to the surface was performed as described by Oliveira and colleagues [48
] with 0.4 nmol sdAb per μmol of total lipids and it resulted in high coupling efficiencies. Among various conditions of the coupling reaction tested, we also tested higher sdAb-to-lipid ratios, but this resulted in precipitation of the liposomes. The fraction of uncoupled sdAb in the liposome suspension was negligible and it did not apparently interfere with binding on cells.
Confocal microscopy Rh4-FR4wt cells incubated with the fluorescent FGFR4-targeting liposomes showed a very specific internalization, represented by dot-like structures within the cells, which were absent in Rh4-FR4ko cells. The images were taken after 2 h of incubation, indicating a rather fast internalization process which can represent an advantage for a drug delivery platform to highly vascularized tumors.
In vitro cell assays are not ideal to predict and compare the in vivo therapeutic effects of drug-loaded targeting nanovesicles. Nonetheless, to verify if the increased binding and internalization observed by fluorescence analysis could translate into an increased activity of targeted VCR-loaded liposomes, we incubated RMS Rh4-FR4wt and Rh4-FR4ko cells with increasing concentrations of targeted liposomes and control L-mCh. We were not able to see significant differences in IC50 between the targeted liposomes and control, or between Rh4-FR4wt and Rh4-FR4ko cells, even after washing off the liposomes after 1 h or 2 h of incubation, to prevent any unspecific drug release during the three days of cultivation. The reasons for the lack of difference in activity between targeted and non-targeted liposomes are not clear. One hypothesis is that it might be due to unspecific binding of the liposomes to cell culture plates causing the release of the cytostatic content. Therefore, the therapeutic potential of FGFR4-targeted drug delivery to RMS needs to finally be evaluated in an RMS in vivo model.
Importantly, we were able to verify the selective cell-mediated cytotoxicity of sdAb-based FGFR4 CAR T cells towards Rh4-FR4wt. Although we observed some differences in cytotoxic efficiencies between three CD8+ T cell donors, all FGFR4-CAR Ts showed the same specific trend. Real-time cell analysis represents an elegant tool to monitor the cytotoxic potential of CAR Ts and revealed no or lower effects of FGFR CAR Ts on Rh4-FR4ko, comparable to that of control CD19 CAR Ts. We believe that the immune-based treatment of RMS with FGFR4 CAR Ts holds promising potential, since RMS tumors display aberrantly high FGFR4 expression compared to healthy tissues [42
]. It has been shown that high antigen densities above a certain threshold level are required for effective CAR T cell activation, offering a therapeutic window for RMS treatment [49
]. Further studies will be required to test FGFR4 CAR Ts efficiency in a RMS in vivo model.
4. Materials and Methods
4.1. Plasmids and Cloning
For recombinant protein expression, sdAb encoding sequences on the pHEN2 phagemid vector were PCR amplified with SapI-introducing primers for FX cloning [51
] into pSB_initC (kindly provided by M. Seeger lab, University of Zurich, Zurich, Switzerland). The expression vector harbors a ccdB suicide cassette, a C-terminal cysteine followed by Myc-tag and 6xHis-tag. Successful cloning of sdAb sequences replaced ccdB and the constructs were amplified in E. coli
MC1061. CAR T cell constructs were generated with the A8 sdAb sequence and were cloned by ligation into the pTRIP-BFP-2a-scFvCD19-myc-41BB-CD3ζ-SBP with the substitution of the scFvCD19 by A8 (pTRIP-BFP-2a-vHH-FGFR4-myc-41BB-CD3ζ-SBP). The pTRIP-BFP-2a-scFvCD19-myc-41BB-CD3ζ-SBP was previously generated by gene synthesis of the sequence composed of: Single peptide CD8α/Single-chain variable fragment against CD19/Myc tag/CD8α hinge and transmembrane domain/Stimulatory domains 41BB and CD3ζ/Streptavidin binding peptide (SBP). This gene was cloned into the pTRIP-SFFV-tagBFP-2A kindly provided by Nicolas Manel (Institut Curie, Paris, France) [52
4.2. Cell Lines
The cell lines Rh4 (kindly provided by Peter Houghton, Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA), Rh30, HEK293ft, HEK293T (purchased from ATCC, LGC Standards S.a.r.l, Molsheim, France) were maintained in DMEM supplemented with 10% FBS (both Sigma-Aldrich, Buchs, Switzerland), 2 mM L-glutamine and 100 U/mL penicillin/streptomycin (both from Thermo Fisher Scientific, Illkirch, France) at 37 °C in 5% CO2
. RMS cell lines were tested and authenticated by cell line typing analysis (STR profiling) in 2014/2015 and positively matched [53
]. All cell lines tested negative for mycoplasma.
4.3. Generation of CRISPR/Cas9 FGFR4 Knockout Cells
Rh4 FGFR4 knockout clones were generated via CRISPR/Cas9 technology. Complementary single-strand oligonucleotides encoding the sgRNA sequence for FGFR4 knockout (TTGCACATAGGGGAAACCGT) were annealed and cloned into the lentiCRISPRv2 puro vector (#98290, Addgene) via Esp3I (ER0451, Thermo Fisher Scientific, Illkirch, France) restriction and T4 ligation (15224017, Thermo Fisher Scientific, Illkirch, France). Lentiviral vectors were produced in HEK293T cells. The cells were transiently transfected with pMDL, pREV, pVSV-G and the lentiCRISPRv2-sgFR4Ex14 using JetPrime (Polyplus Transfection, Illkirch-Graffenstaden, France). After 24 h, medium was replaced, and virus supernatant was harvested after additional 48 h. The supernatant was filtered, 20-fold concentrated (Amicon Ultra 15, Merck Millipore, Schaffhausen, Switzerland; 4000× g, 15 min) and stored at −80 °C. Transduction of RMS cells was performed with concentrated viral supernatant in the presence of 10 µg/mL polybrene (Merck Millipore, Schaffhausen, Switzerland). After 24 h, medium was changed and puromycin selection at 1 µg/mL was started after 72 h and carried out for 7 days. Single-cell cloning was performed with selected cells on 96-well plates and the FGFR4 knockout was confirmed on protein level by Western blotting. All experiments were performed with the knockout clone #8.
4.4. Production of Lentiviral Vector for CAR T Cell Construction
Lentivirus particles were produced by co-transfection of the plasmid containing the genes of interest (BFP-2a-scFvCD19/VHH-FGFR4-myc-41BB-CD3ζ-SBP), the packaging plasmid psPAX2 and envelop plasmid pVSV-G into HEK293ft using the polyethyleneimine (PEI) precipitation protocol. The cells were incubated at 37 °C with 5% CO2 and the supernatant was harvested after 48 h and 72 h, pooled and filtered using a 0.45 μm filter. To concentrate the lentivirus particles, 20% sucrose in PBS was applied to the filtered supernatant followed by centrifugation at 100,000× g for 1.5 h at 4 °C. The pellet was recovered in 1 mL of freezing medium (DMEM complete medium + 0.1 mM β-mercaptoethanol (Gibco, Thermo Fisher Scientific, Illkirch, France) and 1 mM HEPES (Gibco, Thermo Fisher Scientific, Illkirch, France) and stored at −80 °C until use. Lentivirus titer was determined by flow cytometry through the detection of fluorescent protein (mTagBFP) in HEK293ft cells 72 h after transduction.
4.5. T Cell Isolation and Transduction
Peripheral blood mononuclear cells (PBMCs) were recovered using the density gradient Lymphoprep (StemCells, Grenoble, France). CD8+ T cells were isolated by negative selection using the CD8+ T cell human isolation kit (Miltenyi Biotec, Paris, France). Isolated CD8+ T cells were then cultured in X-VIVO medium (Lonza, Colmar, France) supplemented with 50 μM of β-mercaptoethanol (Merck Millipore, Schaffhausen, Switzerland) and 5% human serum (Merck Millipore, Schaffhausen, Switzerland) and activated using human T-activator CD3/CD28 Dynabeads (Gibco, Thermo Fisher Scientific, Illkirch, France). Approximately 24 h after T cell activation, the T cells were transduced with lentiviral particles mixed with 4 μg/mL of polybrene (Merck Millipore, Schaffhausen, Switzerland) at an MOI of 5 or higher. Two days after, the medium was exchanged and replaced by fresh medium supplemented with 5 ng/mL recombinant human interleukin-2 (IL2; R&D Biosystem, Bio-Techne SAS, Noyal Châtillon sur Seiche, France). The transduction efficiency was evaluated at day 6 or 7 after transduction through the detection of mTagBFP expressing cells using flow cytometry. The healthy adult blood donors (Saint-Louis Etablissement Français du sang (EFS) or Saint-Antoine Crozatier EFS at Paris, France) consented to provide their blood for research purposes.
4.6. Phage Display Selection
Screening for FGFR4 binding sdAb was performed with biotinylated extracellular FGFR4 (G&P Biosciences, Santa Clara, CA, USA) in native condition as described [54
] using Nali-H1 library [36
] composed of 3 × 109
synthetic humanized VHH and Gimli library [37
] composed of 1.6 × 109
synthetic human VH.
4.7. Protein Expression and Purification
Periplasmic expression of sdAb was performed in E. coli MC1061 harboring the pSB_init vector enabling protein production with a C-terminal cysteine and 6xHis-tag. A 20 mL overnight pre-culture grown in Terrific Broth medium (25 µg/mL Chloramphenicol) was diluted in 2000 mL fresh medium and grown at 37 °C for 2 h. The temperature was then reduced to 25 °C and after 1 h protein expression was induced with 0.02% L-arabinose. The bacterial culture was grown overnight at 25 °C and cells were harvested by centrifugation (12,000× g, 15 min). Periplasmic protein extraction was performed with the osmotic shock method. The cells were resuspended with 50 mL lysis buffer 1 (50 mM Tris/HCl, pH 8.0, 20% sucrose, 0.5 mM EDTA, 5 μg/mL lysozyme, 2 mM DTT) and incubated for 30 min on ice. After the addition of ice-cold lysis buffer 2 (PBS, pH 7.5, 1 mM MgCl2, 2 mM DTT), the cell debris was harvested by centrifugation (3800× g, 15 min) and the protein-containing supernatant was supplemented with a final concentration of 10 mM imidazole. Ten mL of Co2+-beads slurry (HisPur Cobalt Resin, Thermo Fisher Scientific, Illkirch, France) were washed with wash buffer (PBS, pH 7.5, 30 mM imidazole, 2 mM DTT) and the supernatant was added to the beads. After an incubation of 1 h at 4 °C, the beads were washed with 20 mL wash buffer and bound protein was eluted with 20 mL elution buffer (PBS, pH 7.5, 300 mM imidazole, 2 mM DTT). Prior size exclusion chromatography (SEC), with a Sepax SRT-10C SEC100 column (Sepax Technologies, Newark, DE, USA) equilibrated with PBS, pH 7.5, 2 mM DTT, the protein elution was dialyzed overnight into PBS, pH 7.5, 2 mM DTT and concentrated via spin filter centrifugation (Amicon Ultra 15, 3 kDa, Merck Millipore, Schaffhausen, Switzerland).
4.8. Flow Cytometry
Binding validation of selected phages, recombinant sdAb and decorated liposomes was performed on Rh4-FR4wt and Rh4-FR4ko cells. The specificity of selected phage clones binding to FGFR4 was determined by flow cytometry in 96-well plates (BD Biosciences, Le Pont de Claix, France). Cell surface staining of Rh4-FR4wt or Rh4-FR4ko cells was performed on ice in PBS supplemented with 1% FBS. Eighty µL phages + 20 µL PBS/1% milk were incubated on 1 × 105
cells for 1 h on ice. After 2 washes in PBS, phage binding was detected by a 1:250 dilution of anti-M13 antibody (27-9420-01; GE healthcare, Buc, France) for 1 h on ice followed by a 1:400 dilution of A488-conjugated anti-Mouse antibody (715-545-151; Jackson ImmunoResearch, Europe Ltd., Ely, UK) for 45 min. Samples were analyzed after two washes by flow cytometry on a MACSQuant cytometer (Miltenyi, Biotec, Paris, France) and results were analyzed with FlowJo software (BD Biosciences, Le Pont de Claix, France). Phages displaying anti-mCherry sdAb were used as a negative control [36
]. For binding tests of recombinant sdAb, cells were detached with Accutase (Stemcell Technologies, Grenoble, France) and washed with PBS. All following steps were performed on ice: 4 × 105
cells were incubated with sdAb concentrations of 30 µg/mL for 1 h, washed once with PBS and incubated for an additional 30 min with anti-6xHis-tag FITC-labeled antibody (LS‑C57341, LSBioscience, LabForce. Muttenz, Switzerlnad, diluted 1:10). The cells were washed once more with PBS and analyzed. Targeting liposomes were added at 0.5 mM final lipid concentration in complete medium to cells in 96-well plates and incubated for 2 h at 37 °C and 5% CO2
. The cells were washed twice with PBS and detached with Accutase. All flow cytometry measurements were performed with Fortessa flow cytometer (BD Biosciences, Allschwil, Switzerland) and the data were analyzed using FlowJoTM
10.4.1 software (Becton, Dickinson & Company, Franklin Lakes, NJ, USA). Statistical analysis was performed with GraphPad Prism, version 8 (San Diego, CA, USA). The statistical difference was assessed by paired t-test and p
≤ 0.05 was considered as statistically significant.
4.9. FGFR4 Activation Assay
To test the effect of sdAb on FGFR4 activation, 6 × 104 Rh30-FR4wt and Rh30-FR4ko cells were plated on 24-well plates. The next day, sdAb were added at 10 µM concentrations to the cells in FBS-free medium and incubated for 1 h at 37 °C prior to stimulation with 50 nM recombinant human FGF19 (Peprotech, Lubio science, Zurich, Switzerland) for 10 min. Cells were immediately washed with ice-cold PBS and lysed in Tris/RIPA buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% Na-Deoxycholate, 0.1% SDS, 1 mM EGTA, with standard protease and phosphatase inhibitors). Total cell extracts were then analyzed by Western blotting.
4.10. Western Blotting
SDS-PAGE samples were separated on 4–12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific, Illkirch, France) and blotted on Trans-Blot Turbo Transfer Blot membranes (Biorad, Cressier, Switzerland). After blocking the membranes with blocking buffer (5% milk/TBST) for 1 h at room temperature, the primary antibody was added at a 1:1000 dilution and incubated overnight at 4 °C. The secondary HRP-conjugated antibody was diluted 1:10,000 in blocking buffer and added to the washed membrane for 1 h at room temperature. Chemiluminescence was detected after incubation with AmershamTM ECLTM detection reagent (GE Healthcare, Opfikon, Switzerland) or SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Illkirch, France) in a ChemiDocTM Touch Imaging System (BioRad, Cressier, Switzerland). Primary antibodies used were p44/42 MAPK ERK1/2 (#9102), phospho-p44/42 MAPK Thr202/Tyr204 (#9101), β-Tubulin D3U1W (#86298), FGF Receptor 1 D8E4 (#9740) (all from Cell Signaling Technology, BioConcept, Allschwil, Switzerland), FGF Receptor 2 C-17 (sc-122), FGF Receptor 3 B9 (sc-13121) and FGF Receptor 4 A-10 (sc-136988) (all from Santa Cruz Biotechnology, LabForce, Muttenz, Switzerland). Secondary antibodies were anti-rabbit IgG (#7074, Cell Signaling Technology, BioConcept, Allschwil, Switzerland) and anti-mouse IgG (#7076, Cell Signaling Technology, BioConcept, Allschwil, Switzerland).
4.11. Surface Plasmon Resonance Spectroscopy
Single-cycle kinetics analysis was performed with the BIAcore T200 instrument (GE Healthcare, Opfikon, Switzerland) on CMD200M sensor chips (XanTec bioanalytics GmbH) activated with a mixture of 300 mM NHS (N-hydroxysuccinimide) and 50 mM EDC (N-ethyl-N’-(dimethylaminopropyl) carbodiimide). Recombinant FGFR1, FGFR2, FGFR3 and FGFR4 (G&P Biosciences) were immobilized on the activated biosensors (800 to 12,000 RU; 1 RU = 1 pg/mm2) followed by a blocking step with 1 M ethanolamine. One flow channel per chip was used as a reference to provide background corrections. The sdAb were injected at 5 different concentrations followed by a dissociation phase. All injections and washing steps were performed with TBST buffer. Koff-rates were determined from a final dissociation step after the last injection. The measurements with FGFR4 were performed for each sdAb on freshly immobilized protein due to strong binding and incomplete dissociation from the surface. The immobilization flow rate was 5 μL/min and binding studies were performed at 30 μL/min. Binding parameters were determined with the heterogeneous ligand model fit of the BIAevaluation software.
4.12. Preparation of Fluorescently Labelled VCR-Loaded Liposomes
The production of liposomes and vincristine (VCR) loading was performed as described [21
], with minor modifications. Liposomes were produced with the film-hydration/extrusion method with egg sphingomyelin (Lipoid GmbH, Ludwigshafen am Rhein, Germany), cholesterol (Sigma Aldrich, Buchs, Switzerland), PEG-ceramide (N
-palmitoyl-sphingosine-1-[succinyl [methoxyPEG-2000]]), DSPE-PEG-maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N
-[maleimide (polyethylene glycol)-2000]) (both Avanti Polar Lipids, Alabaster, AL, USA) and DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine Iodide) (Thermo Fisher Scientific, Illkirch, France) at a ratio of 49.8:45:4:1:0.2 mol%, respectively. The lipid film was hydrated with citrate buffer (250 mM, pH 3) resulting in a multilamellar liposomal dispersion having 70 mM of total lipid concentration. Next, six freeze-thaw cycles and ten extrusion steps with a LIPEX®
Thermobarrel extruder (Evonik Nutrition and Care GmbH, Essen, Germany) and a 100 nm pore-size polycarbonate membrane (Whatman, Maidstone, UK) were performed. A transmembrane pH gradient was generated via gel exclusion chromatography with PD MidiTrap™ Sephadex G-25 columns (GE Healthcare, Opfikon, Switzerland). The columns were conditioned with coupling buffer (PBS, pH 7.0) and the eluted liposome suspensions (14 mM) were used for VCR encapsulation. For a molar drug-to-lipid ratio of 0.05, 1 mL of liposomes were mixed with 1 mL of 0.7 mM VCR (VincristineTeva, Teva Pharma AG, Basel, Switzerland) diluted in coupling buffer and incubated for 1 h at 65 °C. Non-encapsulated VCR was removed via spin filter centrifugation (Amicon Ultra 0.5, 100 kDa, Merck Millipore, Schaffhausen, Switzerland). Final VCR-loaded liposomes preparations had a total lipid concentration of 11.2 mM.
4.13. Decoration of Liposomes with sdAb
For coupling of the sdAb to the liposomal surface, the buffer was exchanged to coupling buffer (PBS, pH 7.0) with PD MiniTrap™ Sephadex G-25 columns (GE Healthcare, Opfikon, Switzerland). A sdAb to lipid ratio of 0.4 nmol/μmol was chosen for the reaction, resulting in approximately 30 sdAb per liposome, as described previously [48
]. The reaction was incubated overnight at 4 °C and non-coupled sdAb were removed by two steps of washing and filtration via spin filter centrifugation (Amicon Ultra 0.5, 100 kDa, Merck Millipore, Schaffhausen, Switzerland). The mean diameter and polydispersity index (PDI) of liposomes were measured by dynamic light scattering (Litesizer 500, Buchs, Switzerland). To estimate the amount of sdAb coupled to the liposomes, gel electrophoresis was performed with labelled liposomes and defined amounts of the corresponding sdAb under denaturing and reducing conditions. Sample separation, Western blotting and imaging were performed as described above with anti-6xHis-tag antibody (ab 18184, Abcam, Cambridge, UK).
4.14. VCR Quantification
Quantification of VCR concentrations was performed via HPLC (Ultimate 3000 HPLC system equipped with a DAD-3000 diode array detector, Thermo Fisher Scientific, Illkirch, France) with an RP-18 (5 μm, 4.6 × 250 mm) LiChrospher®
100 column (Merck Millipore, Schaffhausen, Switzerland), optimizing a previously reported method [21
]. A calibration curve for VCR ranging from 890 µg/mL to 13.9 µg/mL was prepared and liposome samples were disrupted with methanol for analysis. Doxorubicin was mixed to all samples to a final concentration of 50 µg/mL, serving as an internal standard. A di-potassium phosphate buffer (50 mM, pH 3.2) was used as mobile phase (68%) with a mixture of acetonitrile/UPW 90/10 (v/v
; 32%) for 30 min at a flow rate of 1.5 mL/min. For each sample, a volume of 20 μL was injected. VCR and doxorubicin were detected at λ = 230 nm. Drug-loading efficiency was determined by analyzing VCR concentrations in the spin-filter purified liposome suspension and the aqueous flow-through. The encapsulation efficiency represented the percentage of VCR in the liposome suspension compared to the combined amount of VCR from filtered liposomes and flow-through.
4.15. Confocal Microscopy
Detection of binding and internalization of fluorescent liposomes was performed on Rh4 wildtype and Rh4-FGFR4-knockout cells via confocal laser scanning microscopy (CLSM-Leica SP8 inverse, Heerbrugg, Switzerland). An amount of 40,000 cells were seeded in a four-well microscopy slide (Falcon™ Chambered Cell Culture Slides, Fisher Scientific, Illkirch, France). The next day, targeted or control liposomes were added to the cells at a final lipid concentration of 3 mM, and incubated for 2 h at 37 °C and 5% CO2. The wells were then washed twice with PBS and the cells were fixed for 15 min with 4% formaldehyde solution. After two further washing steps with PBS, the slides were separated from the chamber case and mounted with DAPI-containing medium (VECTASHIELD® Hardset Antifade Mounting Medium with Phalloidin, Vector Laboratories, Adipogen AG, Liestal, Switzerland). Microscopy imaging was performed with 63x objective (HC PL APO CS2 63x/1.30) and the lasers Diode405 and Diode638 for DAPI and DiR excitation, respectively. All images were processed with ImageJ (v1.52s).
4.16. CAR T Cell Cytotoxicity Assays
Two methods were used to evaluate the cytotoxicity of T cells toward RMS cells. For the bioluminescence assay, Rh4-FR4wt and Rh4-FR4ko cells were transduced with lentiviral particles to express both mTagBFP and Red Firefly luciferase using a P2A fusion. In addition, a fluorogenic reporter YFAST [55
] fused to puromycin resistance gene was expressed using an EF1 promoter (BFP-P2A-Luciferase-pEF1-YAST-Puromycin). Briefly, 4000 target cells were plated in a 96-well ViewPlate Black (Perkin-Elmer, Villebon-sur-Yvette, France) in complete DMEM medium and effector cells (CD8+
T cells) were added the next day at the indicated effector to target (E:T) ratios in X-VIVO medium (2-fold volume compared to DMEM). After approximately 72 h of incubation at 37 °C and 5% CO2
, the wells were washed twice with PBS and 1–2 mg/mL of luciferin substrate (Perkin Elmer, Villebon-sur-Yvette, France) in PBS was added for 10 min (37 °C) prior to luminescence measurement with FLUOstar OPTIMA (BMG LabTech, Champigny-sur-Marne, France). The percentage of cell survival was calculated by taking the luminescence values for each point and dividing it by the highest value of luminescence obtained. Real-time cell death measurements were performed with the xCELLigence Real-Time Analyzer System (ACEA Biosciences, San Diego, CA, USA). Briefly, 10,000 target cells were plated in a 16-well E-plate (ACEA Biosciences, San Diego, CA, USA) in complete DMEM medium and the next day the effector cells were added at indicated E:T ratios in X-VIVO medium (2-fold volume compared to DMEM). Cell index (relative impedance) was monitored in real-time every 15 min for about four days at 37 °C and 5% CO2