Biomedicine & Pharmacotherapy 150 (2022) 112940 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha A diphtheria toxin-based nanoparticle achieves specific cytotoxic effect on CXCR4+ lymphoma cells without toxicity in immunocompromised and immunocompetent mice Aïda Falgàs a, b, c, Annabel Garcia-León a, b, Yáiza Núñez a, b, Naroa Serna c, d, e, Laura Sánchez-Garcia c, d, e, Ugutz Unzueta a, b, c, d, Eric Voltà-Durán c, d, e, Marc Aragó a, Patricia Álamo a, b, c, Lorena Alba-Castellón a, b, Jorge Sierra a, b, f, *, Alberto Gallardo g, Antonio Villaverde c, d, e, Esther Vázquez c, d, e, **, Ramon Mangues a, b, c, ***, Isolda Casanova a, b, c a Biomedical Research Institute Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona 08025, Spain b Josep Carreras Leukaemia Research Institute (IJC), Barcelona 08916, Spain c CIBER de Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), Madrid 28029, Spain d Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Barcelona 08193, Spain e Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Barcelona 08193, Spain f Department of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona 08025, Spain g Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona 08025, Spain A R T I C L E I N F O A B S T R A C T Keywords: High rates of relapsed and refractory diffuse large B-cell lymphoma (DLBCL) patients and life-threatening side Targeted-drug delivery effects associated with immunochemotherapy make an urgent need to develop new therapies for DLBCL patients. Nanoparticle Immunotoxins seem very potent anticancer therapies but their use is limited because of their high toxicity. Diphtheria toxin Accordingly, the self-assembling polypeptidic nanoparticle, T22-DITOX-H6, incorporating the diphtheria toxin CXCR4 receptor DLBCL and targeted to CXCR4 receptor, which is overexpressed in DLBCL cells, could offer a new strategy to selectively Multivalency eliminate CXCR4+ DLBCL cells without adverse effects. In these terms, our work demonstrated that T22-DITOX- H6 showed high specific cytotoxicity towards CXCR4+ DLBCL cells at the low nanomolar range, which was dependent on caspase-3 cleavage, PARP activation and an increase of cells in early/late apoptosis. Repeated nanoparticle administration induced antineoplastic effect, in vivo and ex vivo, in a disseminated immunocom­ promised mouse model generated by intravenous injection of human luminescent CXCR4+ DLBCL cells. More­ over, T22-DITOX-H6 inhibited tumor growth in a subcutaneous immunocompetent mouse model bearing mouse CXCR4+ lymphoma cells in the absence of alterations in the hemogram, liver or kidney injury markers or on- target or off-target organ histology. Thus, T22-DITOX-H6 demonstrates a selective cytotoxicity towards CXCR4+ DLBCL cells without the induction of toxicity in non-lymphoma infiltrated organs nor hematologic toxicity. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; AUC, area under the curve; BLI, bioluminescence imaging; BM, bone marrow; CLS, capillary leak syndrome; DLBCL, diffuse large B-cell lymphoma; FDA, Food and Drug Administration; H&E, hematoxylin and eosin; IHC, immunohistochemistry; IV, intravenous; LN, lymph node; MFI, mean fluorescence intensity; PARP, poly (ADP-ribose) polymerase; PLT, platelets; RBC, red blood cells; SC, subcutaneous; WBC, white blood cells. * Correspondence to: Biomedical Research Institute Sant Pau (IIB-Sant Pau), Josep Carreras Leukaemia Research Institute (IJC) and Department of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. ** Correspondence to: Department of Genetics and Microbiology (Universitat Autònoma de Barcelona) and Institute of Biotechnology and Biomedicine (IBB) , Barcelona, and CIBER de Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain. *** Correspondence to: Biomedical Research Institute Sant Pau (IIB-Sant Pau) and Josep Carreras Leukaemia Research Institute (IJC), Barcelona, and CIBER de Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain. E-mail addresses: jsierra@santpau.cat (J. Sierra), esther.vazquez@uab.cat (E. Vázquez), rmangues@santpau.cat (R. Mangues). https://doi.org/10.1016/j.biopha.2022.112940 Received 21 February 2022; Received in revised form 27 March 2022; Accepted 6 April 2022 Available online 11 April 2022 0753-3322/© 2022 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 1. Introduction 2.2. Cell culture Diffuse large B cell lymphoma (DLBCL) is the most common subtype Human Toledo and U-2932 DLBCL cell lines were cultured with of non-Hodgkin lymphoma accounting around 30–40% of all cases [1]. RPMI 1640 medium whereas the human SUDHL-2 DLBCL cell line was First-line therapy for these patients combines the monoclonal antibody cultured in IMDM medium. All cell lines were supplemented with 10% anti-CD20 (rituximab) with cyclophosphamide, doxorubicin, vincris­ fetal bovine serum (FBS), 1% glutamine and 100 U/mL penicillin- tine, and prednisone (R-CHOP). Nevertheless, between 40% and 50% of streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). Toledo DLBCL patients relapse or have refractory disease [2,3]. R-CHOP is the cell line was purchased from the American Type Culture Collection common treatment for DLBCL and combines Rituximab immunotherapy (ATCC, Manassas, Virginia, USA) and U-2932 cell line from the German with chemotherapy agents that damage the DNA or Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, microtubule-destabilizing agents that kill dividing cells but spare Germany). Finally, SUDHL-2 cell line was kindly provided by Dr L. non-dividing cells [4]. Moreover, chemotherapy passively diffuses Pasqualucci (Columbia University, NY, USA) and its cell line identity through all organs killing both tumor cells and normal cells. For this was verified at genomics and transcriptomics platform at IIB-Sant Pau. reason, patients can experience severe side effects, including As reported in previous works [21,22], Toledo and U-2932 cells life-threatening effects such as bone marrow (BM) myelosupression, express high membrane CXCR4 levels whereas SUDHL-2 does not. The cardiotoxicity or neurotoxicity [5–8]. Thus, targeted therapeutic ap­ SUDHL-2 cell line was transfected by nucleofection (Nucleofector TM 2b proaches are needed aiming to increase the cytotoxicity of lymphoma Device, Lonza, Basel, Switzerland) with the pCXCR4 plasmid, kindly cells whereas reducing the off-target toxicity. provided by Dr. Jun Komano (Osaka Prefectural Institute of Public Bacterial toxins are increasingly used as potent anticancer agents due Health, Osaka, Japan) and CXCR4 positive cells were selected with 0.4 to their high cytotoxicity and they are usually linked to a monoclonal mg/mL of geneticin (Thermo Fisher Scientific), resulting a new cell line antibody (as immunotoxins) that confers them the specificity towards a named CXCR4+ SUDHL-2. Furthermore, the Toledo cell line was target molecule in order to reduce the unspecific toxicity. Three nucleofected with the Luciferase gene (pPK-CMV- F3, Promokine, TE immunotoxins have been approved by the Food and Drug Administra­ Huissen, The Netherlands) and the resulted Toledo-Luci cells were tion (FDA), denileukin diftitox, moxetumomab pasudotox and selected with 0.2 mg/mL of geneticin in order to achieve stable clones. tagraxofusp-erzs, containing a monoclonal antibody conjugated to a Additionally, the murine A-20 lymphoma cell line was cultured with toxin payload, either the diphtheria or the Pseudomonas aeruginosa toxin RPMI 1640 medium, 10% FBS, 1% glutamine, 100 U/mL penicillin- [9–12]. However, the possible drug leakage in the bloodstream and the streptomycin, 0.05 mM 2-mercaptoethanol (Sigma-Aldrich, Saint small amount of the injected dose (<1%) reaching the tumor can limit Louis, MO, USA) and 10 mM Hepes (Thermo Fisher Scientific). A-20 cell the development of immunotoxins [13,14]. line was purchased from the ATCC. Taking into account the risks and limitations of immunotoxins, we developed a new strategy based on a self-assembling toxin-based 2.3. Cell viability assays nanoparticle named T22-DITOX-H6, with a size over 30 nm. The nanoparticle is composed of monomers, each being constructed by a Cell viability assays were used to assess the nanoparticle cytotoxicity single polypeptide chain that includes the translocation and catalytic in human and murine lymphoma cell lines. Lymphoma cells were domains of the diphtheria toxin. Moreover, this nanoparticle construct seeded, in 96-well plates, from 2.5⋅105 to 3.5⋅105cells/mL depending on includes a hexa-histidine tail (H6) for nanoparticle purification by af­ their growth rate. After 24 h, cells were exposed to different concen­ finity chromatography and the T22 ligand for the targeting to CXCR4 trations of T22-DITOX-H6 or buffer (166 mM NaCO3H, pH=8). More­ receptor. Our group reported that CXCR4 is a good target for DLBCL over, for competition assays, aiming to test if nanoparticle entrance in patients since CXCR4 overexpression is found in around 30–50% of cancer cells is mediated by the CXCR4 receptor, cells were pretreated DLBCL patient samples [15,16]. Moreover, CXCR4 overexpression in with the CXCR4 antagonist AMD3100 (Sigma-Aldrich) 1 h before the DLBCL patients is involved in relapse and resistance to R-CHOP and nanoparticle addition (10 AMD3100: 1 T22-DITOX-H6 concentration confers them poor progression-free survival and overall survival ratio). Thus, cancer cell exposure to AMD3100 blocks its membrane [15–18]. Furthermore, the nanoparticle includes a natural CXCR4 receptors, leading to the inhibition of the internalization of the furin-cleavage site between the T22 ligand and the toxin domains for the CXCR4-targeted nanotoxin. Evaluation of cell death caspase- release of the toxin once internalized into target cells, as well as another dependence was performed adding z-VAD-FMK pancaspase inhibitor furin-cleavage site that separates the catalytic domain from the trans­ (Adooq Bioscience, Irvine, CA, USA) at 100 μM 1 h prior nanoparticle location domain, allowing to exert its cytotoxic function into target cells. exposition. Once into the cytoplasm, the catalytic domain inactivates protein syn­ Then, the colorimetric cell proliferation kit reagent (Roche Di­ thesis to induce cell death [19,20]. agnostics, Basel, Switzerland) was added after 48 h of nanoparticle In this work, we aim at evaluating the specific in vitro cytotoxic effect exposure and, after 4 h of incubation, cell viability was quantified by of T22-DITOX-H6 in human and murine CXCR4+ DLBCL cells as well as measuring the absorbance at 492 nm wavelength using a FLUOstar the in vivo anti-lymphoma effect and on-target as well as off-target OPTIMA spectrophotometer (BMG Labtech, Ortenberg, Germany). Re­ toxicity using both xenograft immunocompromised and immunocom­ sults were expressed as percentage of cell viability in relation to the petent mice. Thus, we expect that the combination of our compact and corresponding vehicle control (negative controls, 100% of cell viability), multivalent T22-DITOX-H6 nanoparticle, with its active targeting to e.g. cytotoxicity assays testing T22-DITOX-H6 alone, the vehicle control CXCR4-overexpressing lymphoma cells and the potent mechanism of was the nanoparticle buffer (166 mM NaCO3H, pH=8); in competition action of diphtheria toxin, will make for an effective and safe strategy to assays the negative control was the AMD3100 together with the nano­ eliminate CXCR4+ DLBCL cells. particle buffer and in cell death caspase-dependence assays, the negative control was zVAD-FMK together with the nanoparticle buffer. 2. Material and methods 2.4. Flow cytometry 2.1. Therapeutic T22-DITOX-H6 nanoparticle construction For determining the membrane CXCR4 expression, cells were firstly The T22-DITOX-H6 nanoarchitecture, production and purification washed with PBS 0.5% BSA. Then, human SUDHL-2 and CXCR4+ were described in our previous work [20]. SUDHL-2 cells were incubated with PE-Cy5 mouse anti-human CXCR4 monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, USA) or PE- 2 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Cy5 Mouse IgG2a isotype control antibody (BD Biosciences) and murine 2.8. Animal maintenance A-20 cells with PE anti-mouse CD184 (CXCR4) antibody (Biolegend, San Diego, CA, USA) or PE Rat IgG2b isotype control antibody (Biolegend). Four-week-old female NOD/SCID mice and seven-week-old female Incubation with antibodies was performed for 20 min at 4ºC followed by BALB/cByJ mice were obtained from Charles River Laboratories (Wil­ another PBS 0.5% BSA wash. Finally, cells were captured by FACS mington, MA, USA). Mice were housed in microisolator units with sterile Calibur flow cytometry (BD Biosciences) and the results were analyzed food and water ad libitum. All procedures were conducted in accordance using the Cell Quest Pro software and expressed as mean fluorescence with the guidelines approved by the institutional animal Ethics Com­ intensity (MFI). mittee of Hospital Sant Pau and performed following the European Union Directive 2010-63-EU for welfare of the laboratory animals. 2.5. Western blot 2.9. In vivo T22-DITOX-H6 antineoplastic effect in Toledo-Luci 5 5 disseminated mouse model Toledo and U-2932 cells were seeded in flasks at 3.5⋅10 and 3.0⋅10 cells/mL, respectively, and treated with buffer or 5 nM of T22-DITOX- After one week of quarantine, eighteen immunocompromised H6 for 24 h and 48 h. For SUDHL-2 and CXCR4+ SUDHL-2 extracts, NODSCID mice were intravenously injected with 20⋅106 Toledo-Luci cells were seeded at 2.5⋅105 and 3.0⋅105 cells/mL, respectively. Cells cells. Three days later, mice were randomized and divided into two were washed twice with PBS and resuspended in lysis buffer (1 M Tris/ groups (n = 9/group), the control group was treated with buffer (166 acetate, 1 M sucrose, 100 mM EDTA, 100 mM EGTA, 10% TritonX-100, mM NaCO3H, pH=8) and the experimental group was treated with 10 μg 100 mM Naorto, 100 mM Naβglycerol, 0.5 M NaF, 100 mM Napyro, 1% T22-DITOX-H6. The treatment administration was performed intrave­ β-mercapto, 100 mM Benzamidine, 1.74 mg/mL PMSF and 2 mg/mL nously three times per week in a total of thirteen doses. In vivo lym­ leupeptin). The cell suspension was sonicated and rested for 20 min on phoma dissemination was monitored using bioluminescence imaging ice and, then, centrifuged for 10 min at 14,000 rpm and 4 ºC. Protein (BLI) as total radiance photons in the IVIS Spectrum (Perkin-Elmer, concentration in supernatant was determined using the Bradford protein Waltham, MA, USA). For that, mice were anesthetized with 3% iso­ assay, according to the manufacturer’s instructions (BioRad). Cell ly­ flurane in oxygen and BLI was captured 5 min after intraperitoneal in­ sates (50 μg) were mixed with LB6X-DTT 3 M, separated using 12–15% jection of firefly D-luciferin (2.25 mg/mouse, Perkin Elmer). In addition, SDS-PAGE and transferred to a nitrocellulose blotting membrane (GE mouse body weight was registered at least twice per week. All mice were Healthcare life sciences). Membranes were blocked with 5% skim milk euthanized the day that the first animal presented relevant signs of in TBST for 2 h at room temperature. Then, membranes containing disease such as lack of mobility or 10% weight loss. At that day, the BLI Toledo and U-2932 cell lysates were incubated with the following pri­ signal from lymphoma-infiltrated organs was quantified ex vivo as mary antibodies: 1:2000 PARP (556494, BD Biosciences), 1:1000 average radiance photons and then all organs were embedded in paraffin caspase-3 (610322, BD Biosciences) and 1:1000 cleaved caspase-3 for further histological analyses. (9661, Cell Signaling Technology, Danvers, MA, USA); SUDHL-2 and CXCR4+ SUDHL-2 membranes were incubated with 1:1000 CXCR4 2.10. In vivo T22-DITOX-H6 antineoplastic effect in A-20 subcutaneous (1009, Prosci, Fort Collins, CO, USA). The loading control antibody used (SC) mouse model was α/β tubulin (2148, Cell Signaling Technology) at 1:1000. Mem­ branes were washed with TBST and then incubated with the appropriate After one week of quarantine, ten immunocompetent BALB/cByJ secondary antibody (1:10,000, Jackson Immune Research). Western blot mice were subcutaneously injected with 5⋅105 A-20 cells. Tumor growth visualization was performed using the SuperSignal West Pico Chemilu­ was monitored twice a week with bilateral caliper measurements minescent Substrate (Thermo Fisher Scientific) and the G:BOX iChemi (width2 x length/2). When tumors reached a volume between 50 and XT Imaging System (Syngene). 100 mm3, mice were randomly divided in control group (n = 5), treated intravenously with 166 mM NaCO3H, pH= 8 buffer, and experimental 2.6. Annexin/PI assay group (n = 4), treated intravenously with 5 μg T22-DITOX-H6. One mouse was excluded from the experiment because of technical problems Toledo and U-2932 cells were seeded at 3.5⋅105 and 3.0⋅105 cells/ during the intravenous injection. Mice were treated three times per mL, respectively, and treated with buffer (166 mM NaCO3H, pH=8) or 5 week until the first mice achieved a tumor volume size of 500 mm3. nM T22-DITOX-H6 for 15 h, 24 h or 48 h. The percentage of cell Then, animals were euthanized and all organs were embedded in viability, early apoptosis and late apoptosis was evaluated using the paraffin for further histological analyses. Annexin V-CF Blue/PI apoptosis detection kit (Abcam, Cambridge, UK) The area under the curve (AUC) of tumor size from buffer and and following the manufacturer’s instructions. Data were analyzed by nanoparticle-treated mice was calculated using the GraphPad Prism 6 MACSQuant analyzer flow cytometry using the MACS Quantify version program. 2.3 software (Miltenyi Biotec, Bergisch Gladbach, North Rhine- Westphalia, Germany). 2.11. Paraffin-embedded cell blocks A-20 cells were paraffin-embedded from the centrifuged cell sus­ 2.7. DAPI staining pension by adding five drops of plasma and thrombin to enmesh the cellular material in a clot. Then, cell clots were placed in a cassette, fixed Lymphoma cells were seeded from 2.5⋅105 to 3.5⋅105cells/mL in 4% paraformaldehyde and paraffin-embedded in a tissue processor depending on their growth rate and treated with buffer (166 mM (Sakura, Tokyo, Japan). NaCO3H, pH=8) or 2.5 nM or 5 nM T22-DITOX-H6 for 48 h. After that time, cells were washed with PBS, incubated with 3.7% para­ 2.12. Hematoxilin and eosin (H&E) and immunohistochemical staining formaldehyde for 10 min at − 20 ºC, washed again and placed on a slide. Finally, these cells were stained with DAPI mounting medium (Thermo Paraffin-embedded organs from immunocompromised and immu­ Fisher Scientific) and visualized using a fluorescence microscope nocompetent mice were analyzed histopathologically (H&E staining) by (Olympus BX53, Olympus). Representative pictures were taken using an two independent observers to assess the possible toxicity. Olympus DP73 digital camera and processed with the cellSens Dimen­ CXCR4 (1:300, Abcam) expression was evaluated in cellular blocks. sion 1.9 software (Olympus) at 1000X magnification. Immunohistochemistry staining was performed in a DAKO Autostainer 3 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Fig. 1. T22-DITOX-H6 specific cytotoxicity in human CXCR4+ DLBCL cell lines. (A) Cell viability assays performed after 48 h of T22-DITOX-H6 incubation at different concentrations (0.05–5.00 nM) in Toledo and U-2932 cells. (B) Competition assays in Toledo and U-2932 cells with or without pretreating with the antagonist of CXCR4 AMD3100 followed by the nanoparticle incubation for 48 h (ratio 10 AMD3100: 1 T22-DITOX-H6). (C-D) CXCR4 determination in SUDHL-2 and CXCR4+ SUDHL-2 cells by flow cytometry and Western blot. In panel D, α/β tubulin antibody is used as endogen control. (E) Cell viability assays performed in SUDHL-2 and CXCR4+ SUDHL-2 cells after 48 h of T22-DITOX-H6 incubation (0.10–5.00 nM). *p ≤ 0.05. Link48 following the manufacturer’s instructions. Representative pic­ (ALT) enzyme activities, as well as creatinine and uric acid levels, using tures were taken using an Olympus DP73 digital camera and processed commercial kits (ASTL ref. 20764949 322; ALTL ref. 20764957 322, with the Olympus CellD Imaging 3.3 software at x400 or x1000 CREJ2 ref. 04810716 190 and UA2 ref. 03183807 190, Roche Di­ magnifications. agnostics) and adapted for a COBAS 6000 autoanalyzer (Roche Diagnostics). 2.13. Hematological toxicity 2.15. Statistical analysis Blood was obtained by intracardiac punction (25G) the day that immunocompetent mice were killed and blood samples were collected All in vitro experiments were performed in triplicate and in vivo ex­ in EDTA-tubes for the hematological analysis using the BC-5000 Vet periments at least in quadruplicates. Numerical data were expressed as (Mindray, Madrid, Spain). This device allowed us to count the number of mean ± standard error. Differences between groups were analyzed white blood cells (WBC), platelets (PLT) and red blood cells (RBC), but using the Mann-Whitney U test and were considered statistically sig­ also the percentage and number of each type of WBC (neutrophils, nificant at p ≤ 0.05. Statistical calculations were performed using SPSS lymphocytes, monocytes, eosinophils and basophils). software v21. 2.14. Hepatic and renal functionality 3. Results Blood collected in EDTA-tubes was centrifuged at 600g for 10 min at The first step was to assess the specific cytotoxic effect of the T22- 4 ºC for obtaining plasma. Then, plasma samples were analyzed for DITOX-H6 therapeutic nanoparticle in human CXCR4+ DLBCL cell determining the aspartate transaminase (AST), alanine transaminase lines. For that, we used two DLBCL cell lines (Toledo and U-2932) which 4 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Fig. 2. Cell death mechanism induced by T22-DITOX-H6 in human CXCR4+ DLBCL cell lines. (A) Cell viability assays, pretreating Toledo and U-2932 cells with 100 μM of z-VAD-FMK pan caspase inhibitor, followed by the incubation of 5 nM of T22-DITOX-H6 for 48 h. (B) Western Blot analysis for the detection of pro- caspase-3, cleaved-caspase-3, full length Poly (ADP-ribose) polymerase (PARP) and cleaved PARP in Toledo and U-2932 cells, treated with buffer or 5 nM T22- DITOX-H6 for 24 h and 48 h. α/β tubulin antibody is used as endogen control. (C) Representative dot-plot from Annexin-PI assay showing the percent of viable cells (V), early apoptotic cells (EA) and late apoptotic cells (LA) in Toledo an U-2932 cells treated with buffer or 5 nM T22-DITOX-H6 at different time points (15 h, 24 h and 48 h) and (D) their respective quantification in triplicates. (E) DAPI staining images (1000X) of Toledo and U-2932 cells treated with buffer or 2.5 nM T22- DITOX-H6 for 48 h. White arrows point out apoptotic bodies. *p ≤ 0.05. 5 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Fig. 3. In vivo antineoplastic effect of T22-DITOX-H6 in a disseminated CXCR4+ DLBCL mouse model. (A) BLI emitted by Toledo-Luci cells from four representative mice of each group (buffer and T22-DITOX-H6) at days 28, 30 and 33. (B) BLI quantification of mice treated with buffer or T22-DITOX-H6 during the follow-up (n = 9/group). (C) Body weight of mice treated with buffer or T22-DITOX-H6 during the follow-up (n = 9/group). *** p < 0.005. BLI: bioluminescence imaging; iv: intravenous. were previously reported to show high CXCR4 membrane expression Figure 1). Also, performing an Annexin/PI assay, we observed a signif­ [21,22]. After the exposure of these CXCR4+ DLBCL cell lines to the icant increase of the percentage of cells in early and late apoptosis after nanoparticle for 48 h, cell viability was reduced in a 15, 24 and 48 h exposure with 5 nM of nanoparticle in CXCR4+ DLBCL concentration-dependent manner reaching 12.4 ± 1.6% and 20.8 ± cells (Fig. 2C). Indeed, the peak of the percentage of early apoptotic cells 3.8% of cell viability in Toledo and U-2932 cells, respectively, at the was reached at 24 h (17.8 ± 1.2% of total cells in Toledo cells and 12.1 maximum concentration of 5 nM (Fig. 1A). ± 1.6% of total cells in U-2932 cells) and subsequently the peak of late As we wanted to determine whether the nanoparticle cytotoxic effect apoptosis was achieved after 48 h (46.7 ± 4.3% of total cells in Toledo was due to its specific entrance through the CXCR4 receptor, Toledo and cells and 25.8 ± 2.2% of total cells in U-2932 cells) (Fig. 2D). In addi­ U-2932 cells were pretreated with the CXCR4 antagonist, AMD3100, tion, we observed, by DAPI staining, the formation of apoptotic bodies in following the nanoparticle addition. Cells treated with both AMD3100 the lymphoma cells treated with T22-DITOX-H6 for 48 h (Fig. 2E). These and nanoparticle reversed significantly the cytotoxicity reaching up to data suggest the induction of apoptosis by T22-DITOX-H6 in CXCR4+ 90.7% of cell viability compared to cells treated with the nanoparticle DLBCL cells. alone in both cell lines (Fig. 1B). Moreover, we used the parental At that point, we evaluated the nanoparticle cytotoxic effect in a SUDHL-2 cell line (CXCR4-) and the CXCR4+ SUDHL-2 cell line, previ­ CXCR4+ DLBCL luminescent disseminated mouse model generated by ously transduced with the CXCR4 receptor, for testing the nanoparticle intravenous injection of Toledo-Luci cells. Three days after cell injec­ cytotoxic effect. After checking the absence of CXCR4 in SUDHL-2 cells tion, NODSCID mice were treated with buffer or 10 μg of T22-DITOX- and its presence in CXCR4+ SUDHL-2 cells by flow cytometry and by H6, three days per week, in a total of thirteen doses. As observed by Western Blot (Fig. 1C-D and Supplementary Figure 1), we demonstrated the bioluminescence (BLI) emitted by Toledo-Luci cells, the lymphoma that T22-DITOX-H6 did not show cytotoxic effect in the negative-CXCR4 was totally disseminated in buffer-treated mice at last days (days 30 and SUDHL-2 cell line (94.4 ± 4.4%) whereas it did in CXCR4+ SUDHL-2 33) compared to mice treated with T22-DITOX-H6 (Fig. 3A). The BLI cells with 57.3 ± 3.8% of cell viability at 5 nM (Fig. 1E). quantification exhibited highly significant differences between mice Furthermore, the cytotoxic effect of the nanoparticle in CXCR4+ treated with buffer and nanoparticle-treated mice, already at day 17 and DLBCL cells was significantly reverted with the pretreatment of the until day 33, point at which the BLI signal was 9.1-fold times reduced in caspase inhibitor z-VAD (2.0-fold in Toledo cells and 1.5-fold in U-2932 mice treated with T22-DITOX-H6 (Fig. 3B). Neither macroscopic alter­ cells) (Fig. 2A). By Western blot, we observed the reduction of pro- ations (not shown) nor loss of mouse weight was found in nanoparticle- caspase-3 together with the induction of cleaved caspase-3 as well as treated mice (Fig. 3C). An ex vivo analysis of lymphoma-infiltrated or­ the presence of cleaved PARP in Toledo and U-2932 cells after 24 and gans at the end of treatment found that nanoparticle-treated animals 48 h incubation with 5 nM T22-DITOX-H6 (Fig. 2B and Supplementary showed a significant reduction of invading lymphoma cells, detected by 6 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Fig. 4. Ex vivo antineoplastic effect of T22-DITOX-H6 in a disseminated CXCR4+ DLBCL mouse model. BLI quantification and representative images of lymphoma- infiltrated organs (cervical LNs, renal LNs, backbone, hindlimbs and cranium) from Toledo-Luci mice treated either with buffer or T22-DITOX-H6 at the end of the experiment. *p ≤ 0.05, ***p ≤ 0.005. BLI: bioluminescence imaging; LNs: lymph nodes. BLI, in cervical and renal LNs (6.6-fold and 3.6-fold, respectively) as well these cells for 48 h to the nanoparticle, their viability was reduced up to as in BM, including backbone, hind limbs and cranium (6.7-fold, 15.4- 38.33 ± 16.4% at 5 nM (Fig. 5C). Moreover, the cytotoxic effect was fold and 2.3-fold, respectively) (Fig. 4). Moreover, no histopatholog­ significantly reverted (90.9 ± 15.9%) when cells were preincubated ical alterations were found in hematoxylin and eosin (H&E) stained with AMD3100 followed by the nanoparticle addition, confirming that tissue sections of liver, spleen, kidneys, lungs and BM in nanoparticle- the T22-DITOX-H6 cytotoxic effect in murine A-20 cells was performed treated mice (Supplementary Figure 2). specifically through the CXCR4 receptor (Fig. 5D). As we reported with Once we demonstrated the potent cytotoxic effect of the nanoparticle human Toledo and U-2932 cells, the incubation with T22-DITOX-H6 for in human CXCR4+ DLBCL cells in vitro and in vivo in immunocompro­ 48 h produced the formation of apoptotic bodies in murine CXCR4+ A- mised mice, next step was to evaluate the effect of the nanoparticle in 20 cells as well (Fig. 5E). murine CXCR4+ lymphoma cells and especially its toxicity (local, sys­ Then, we evaluated the in vivo antitumor effect of the nanoparticle in temic and functional) in immunocompetent mice. First, we determined a SC A-20 mouse model generated in immunocompetent BALB/cByJ that murine A-20 cells expressed high levels of CXCR4 receptor in the mice. When tumors were palpable (day 10), we started administering cell membrane by IHC and flow cytometry (Fig. 5A-B). After exposure of buffer or T22-DITOX-H6 three times per week until the first tumor of any 7 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Fig. 5. T22-DITOX-H6 specific cytotoxicity in a murine CXCR4+ lymphoma cell line. (A-B) CXCR4 determination by immunohistochemistry and flow cytometry in murine A-20 cells. Scale bars: main picture at 50 µm (400X) and the inset at 20 µm (1000X). (C) Cell viability assays measuring the T22-DITOX-H6 cytotoxicity in A- 20 cells at different concentrations of the nanoparticle (0.25–5.00 nM) for 48 h. (D) Competition assays in A-20 cells with or without the pretreatment of the CXCR4 antagonist AMD3100 followed by the nanoparticle incubation for 48 h (ratio 10 AMD3100: 1 T22-DITOX-H6). MFI: mean fluorescence intensity. (E) DAPI staining pictures (1000X) of A-20 cells treated with buffer or 5 nM T22-DITOX-H6 for 48 h. White arrows point out apoptotic bodies. *p ≤ 0.05. experimental animal reached 500 mm3, point at which we stopped the means that our nanoparticle targets and kills murine and human administration (day 20). As a result, nanoparticle administration CXCR4+ lymphoma cells independently of the presence of murine im­ decreased tumor growth along time. At day 17, tumors reached a vol­ mune cells. ume of 242.0 ± 25.9 mm3 in buffer-treated mice, whereas tumor vol­ It has been reported that immunotoxins incorporating the diphtheria ume was significantly reduced in mice treated with the nanoparticle toxin can cause severe side effects. One example is the diphtheria toxin (121.4 ± 29.7 mm3) (Fig. 6A). Analyzing the area under the curve attached to interleukin-2 (denileukin diftitox), a drug approved by the (AUC) from day 14 to the end of the experiment, a significant 46.9% FDA to treat cutaneous T-cell lymphoma; however, it was withdrawn reduction of tumor size was observed in mice treated with T22-DITOX- from the market in 2014 because of severe side effects [23]. Afterwards, H6 compared to control mice (Fig. 6B). In addition, mouse body weight a novel immunotoxin containing diphtheria toxin was generated by was not altered by the nanoparticle administration (Fig. 6C) and no replacing interleukin-2 by interleukin-3 so that they targeted leukemic histopathological alterations were observed in spleen, liver, kidneys and stem cells. The combination of diphtheria toxin and interleukin-3 BM from nanoparticle-treated mice (Fig. 6D). received the name of tagraxofusp-erzs, and it was approved by the Most importantly, we performed an exhaustive toxicity study to FDA in 2018. Despite being in the market, several clinical trials using evaluate the potential clinical translation of the nanoparticle. This tagraxofusp-erzs reported serious adverse events in various hematologic analysis showed a lack of significant differences between control and neoplasms, including capillary leak syndrome (CLS) of grade ≤ 3 and nanoparticle-treated mice regarding the number of WBC, PLT or RBC three cases of fatal CLS especially in a trial for blastic plasmacytoid (Fig. 7A). Moreover, the percentage of cells in each WBC subtype dendritic cell neoplasm (BPDCN). Moreover, across these trials, throm­ (neutrophils, lymphocytes, monocytes, eosinophils and basophils) was bocytopenia was also a significant side effect reaching grade ≤ 4 as well not altered by nanoparticle treatment (Fig. 7B). We also checked the as transaminitis grade ≤ 3.[24]. Additionally, other immunotoxins hepatic function by measuring the AST and ALT levels as well as the containing diphtheria toxin domains that targeted CD3 or GM-CSFR in renal function determining creatinine and uric acid levels. These data hematologic malignancies showed off-target toxicities in phase I/II showed the absence of hepatic and renal damage in mice treated with clinical trials, including CLS, rare immunocompromised host infections the nanoparticle since these parameters remained unaltered (Fig. 7C). or liver injury [25–27]. Therefore, the T22-DITOX-H6 nanoparticle showed a selective cytotoxic In contrast to immunotoxins, the selective diphtheria toxin delivery effect in murine CXCR4+ lymphoma cells without inducing on-target or accomplished by our T22-DITOX-H6 nanoparticle offers a more effective off-target toxicity. selectivity to target cancer cells since the self-assembled T22-DITOX-H6 nanostructure incorporates multimeric T22 ligands for the CXCR4 re­ 4. Discussion ceptor that enhance internalization in target cells compared to the only two ligands displayed by the immunotoxins [28,29]. Importantly, the Actively targeted nanoparticles are promising toxin-delivery sys­ multi-valency based on a large number of ligand moieties in the nano­ tems. The T22-DITOX-H6 therapeutic nanoparticle has demonstrated particle surface, confers super-selectivity to target cells, which means not only a potent cytotoxic effect in vitro in CXCR4+ DLBCL cells at the that T22-DITOX-H6 is able to interact with the CXCR4 receptor only low nanomolar range, but also a high in vivo efficacy by blocking the when its density in the cell membrane is higher than a specific threshold lymphoma dissemination in the immunocompromised mice and [30,31]. As CXCR4+ DLBCL cells usually show extremely higher levels of reducing tumor growth in immunocompetent mice. This observation the receptor than normal hematopoietic cells (140-fold higher) [18], the 8 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Fig. 6. T22-DITOX-H6 anti-tumor effect, and on-target and off-target toxicity in a SC mouse model bearing murine CXCR4+ lymphoma cells. (A) A-20 SC tumor volume from mice treated with buffer (n = 5) or T22-DITOX-H6 (n = 4) during the whole experiment. (B) AUC quantification of tumor volume from 14 to 20 days in buffer and nanoparticle-treated mice. (C) Body weight follow-up of mice bearing A-20 SC tumors, treated with buffer and T22-DITOX-H6. (D) Hematoxylin and eosin staining from on target and off-target organs (spleen, liver, kidneys and BM). Scale bars: 50 µm. *p ≤ 0.05. AUC: area under the curve; BM: bone marrow; iv: intravenous. super-selectivity property conferred by the T22-DITOX-H6 aims to avoid immunocompetent mice did not show hepatic and renal functional or dramatically minimize any possible toxicity in normal cells that ex­ alteration nor hematological on-target toxicity. Although murine and press low or medium membrane CXCR4 levels. human white blood cells, especially monocytes and lymphocytes, ex­ Furthermore, the tight T22-DITOX-H6 construction in a single press CXCR4 [32–34], the expression levels in those cells are much lower polypeptide chain, without any subsequent chemical conjugation, than those in human CXCR4+ DLBCL cell lines, as we described in our potentially avoids the premature release of the toxin in the mouse previous works [21,22]. bloodstream [20]. Consequently, with this notion, we did not observe According to our strategy, other researchers also used nanoparticle any histopathological alteration in non-lymphoma infiltrated organs in drug delivery systems for targeted delivery of diphtheria toxin in breast immunocompromised nor immunocompetent mice treated with the cancer and melanoma, with high efficiency and precision in vitro and in nanoparticle. Most importantly, the administration of T22-DITOX-H6 in vivo [35,36]. In addition, our group has previously demonstrated the 9 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Fig. 7. Blood analysis and hepatic and renal functional evaluation from T22-DITOX-H6-treated mice bearing A-20 SC tumors. (A) Total number of WBC, PLT and RBC in mice treated with buffer or T22-DITOX-H6. (B) Percentage of the different types of WBC in buffer and nanoparticle-treated mice. (C) Measurement of the AST and ALT enzymes as well as creatinine and uric acid waste products for evaluation of hepatic and renal toxicity, respectively, in mice treated with buffer or T22- DITOX-H6. ALT: alanine aminotransferase; AST: aspartate aminotransferase; PLT: platelets; RBC: red blood cells WBC: white blood cells. antitumor effect of T22-DITOX-H6 in CXCR4+ colorectal and lymphoma Declaration of conflicting interests SC mouse models as well as in a disseminated CXCR4+ acute myeloid leukemia (AML) mouse model [20,21,37]. The authors declare the following financial interests/personal re­ As a conclusion, we believe that the T22-DITOX-H6 diphtheria toxin lationships which may be considered as potential competing interests: delivery system that incorporates the natural toxin is capable of The authors declare the following financial interests/personal relation­ inducing cytotoxic killing of both dividing and non-dividing cells, as ships which may be considered as potential competing interests: E.V., R. reported [38]. This effect shows specificity towards CXCR4+ DLBCL M. and A.V. are co-founders of Nanoligent, a company devoted to cells, therefore, sparing the on-target and off-target toxicities showed by develop anticancer drugs based on proteins. U.U., E.V., A.V., R.M. and I. the immunotoxins. Thus, this toxin-based nanoparticle drug could be a C. are cited as inventors in a patent application (EP11382005.4) good strategy to target and kill CXCR4+ lymphoma cells from refractory covering the therapeutic use of T22. All other authors report no conflicts or relapsed DLBCL patients. of interest in this work. CRediT authorship contribution statement Acknowledgments Conception and design: A.F., N.S., L.S.G., U.U., A.V., E.V., I.C. and R. This work was supported by Instituto de Salud Carlos III (ISCIII, Co- M.; Development of methodology: A.F., A.G.L., Y.N., E.V.D., P.A. and L. funding from FEDER) [PI21/00150, PI18/00650, and EU COST Action A.C.; Acquisition of data: A.F., A.G.L., Y.N. and M.A.; Histopathological CA 17 140 to R.M.; FIS PI20/01621, PI17/01246 and RD16/0011/0028 analysis: A.F. and A.G.; Analysis and interpretation of data: A.F., U.U., J. to J.S.; and PI20/00400 to U⋅U.]; Agencia Estatal de Investigación (AEI) S., A.V., E.V., I.C. and R.M.; Drafted the manuscript: A.F.; Review and and Fondo Europeo de Desarrollo Regional (FEDER) (grant BIO2016- revision of manuscript: U.U., A.V., E.V., I.C. and R.M.; Study supervi­ 76063-R, AEI/FEDER, UE) to A.V.; (grant PID2019-105416RB-I00/AEI/ sion: J.S., E.V., I.C. and R.M. 10.13039/501100011033) to E.V.; CIBER-BBN [CB06/01/1031 and 4NanoMets to R.M., VENOM4CANCER to A.V., NANOREMOTE to E.V. and NANOSCAPE to U⋅U.]; AGAUR [2017-SGR-865 to R.M., 2017-SGR- 1395 to J.S. and 2017SGR-229 to A.V.]; Josep Carreras Leukemia 10 A. Falgàs et al. Biomedicine & Pharmacotherapy 150 (2022) 112940 Research Institute [P/AG to R.M.]; La Marató TV3 [201 941-30-31-32 to relapsed/refractory hairy cell leukemia, Leukemia 32 (2018) 1768–1777, https:// doi.org/10.1038/s41375-018-0210-1. J.S. and A.V.]; a grant from the Cellex Foundation, Barcelona [to J.S.]; a [12] C.F. Nobre, M.J. Newman, A. DeLisa, P. Newman, Moxetumomab pasudotox-tdfk grant from the Generalitat de Catalunya CERCA Programme; U.U. is for relapsed/refractory hairy cell leukemia: a review of clinical considerations, supported by Miguel Servet fellowship (CP19/00028) from Instituto de Cancer Chemother. Pharmacol. 84 (2019) 255–263, https://doi.org/10.1007/ Salud Carlos III co-funded by Fondo Social Europeo (ESF investing in s00280-019-03875-6. [13] G. Aruna, Immunotoxins: a review of their use in cancer treatment, J. Stem Cells your future); L.A.C was supported by a postdoctoral fellowship from Regen. Med. 1 (2006) 31–36, https://doi.org/10.46582/jsrm.0101005. 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Young, Dysregulated CXCR4 expression promotes ICTS Nanbiosis Platform for their funding. The bioluminescent follow- lymphoma cell survival and independently predicts disease progression in germinal up of cancer cells and toxicity studies has been performed in the ICTS- center B-cell-like diffuse large B-cell lymphoma, Oncotarget 6 (2015) 5597–5614, https://doi.org/10.18632/oncotarget.3343. 141007 Nanbiosis Platform, using its CIBER-BBN Nanotoxicology Unit [16] M.J. Moreno, R. Bosch, R. Dieguez-Gonzalez, S. Novelli, A. Mozos, A. Gallardo, (http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/). Pro­ M.Á. Pavón, M.V. Céspedes, A. Grañena, M. Alcoceba, O. Blanco, M. Gonzalez- tein production has been partially performed by the ICTS “NANBIOSIS”, Díaz, J. Sierra, R. Mangues, I. Casanova, CXCR4 expression enhances diffuse large B cell lymphoma dissemination and decreases patient survival, J. 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