Received: 22 September 2020 Revised: 22 December 2020 Accepted: 28 December 2020 Published online: 23 February 2021 DOI: 10.1002/ctm2.280 RESEARCH ARTICLE Enforced sialyl-Lewis-X (sLeX) display in E-selectin ligands by exofucosylation is dispensable for CD19-CAR T-cell activity and bone marrow homing Diego Sánchez-Martínez1 Francisco Gutiérrez-Agüera1 Paola Romecin1 Meritxell Vinyoles1 Marta Palomo1 Néstor Tirado1 Samanta Romina Zanetti1 Manel Juan2 Michela Carlet3,4 Irmela Jeremias3,4 Pablo Menéndez1,5,6,7 1 Department of Biomedicine, Josep Carreras Leukemia Research Institute, Graphical Abstract School of Medicine, University of Barcelona, Barcelona, Spain 2Servei d’Immunologia, Hospital Clínic de Barcelona, Barcelona, Spain 3Department of Apoptosis in Hematopoietic Stem Cells, Helmholtz Center Munich, German Center for Environmental Health (HMGU), Munich, Germany 4Department of Pediatrics, Dr von Glyco-engineered enforced sialofucosylation of E-selectin ligands was dispens- Hauner Children’s Hospital, LMU, Munich, Germany able for CD19-CAR T-cell activity and BM homing in multiple xenograft models, 5Department of Biomedicine, School of thus representing an unnecessary strategy for CD19-CAR T-cell therapy Medicine, University of Barcelona, Barcelona, Spain 6 Centro de Investigación Biomédica en Red-Oncología (CIBERONC), Instituto de Salud Carlos III, Madrid, Spain 7 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Correspondence Dr. Pablo Menéndez and Diego Sánchez- Martínez, Department of Biomedicine, Josep Carreras Leukemia Research Insti- tute, School of Medicine, University of Barcelona, Barcelona, Spain. Email: pmenendez@carrerasresearch.org; dsanchez@carrerasresearch.org Clin. Transl. Med. 2021;11:e280. wileyonlinelibrary.com/journal/ctm2 https://doi.org/10.1002/ctm2.280 Received: 22 September 2020 Revised: 22 December 2020 Accepted: 28 December 2020 Published online: 23 February 2021 DOI: 10.1002/ctm2.280 RESEARCH ARTICLE Enforced sialyl-Lewis-X (sLeX) display in E-selectin ligands by exofucosylation is dispensable for CD19-CAR T-cell activity and bone marrow homing Diego Sánchez-Martínez1 Francisco Gutiérrez-Agüera1 Paola Romecin1 Meritxell Vinyoles1 Marta Palomo1 Néstor Tirado1 Samanta Romina Zanetti1 Manel Juan2 Michela Carlet3,4 Irmela Jeremias3,4 Pablo Menéndez1,5,6,7 1 Department of Biomedicine, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Barcelona, Spain 2 Servei d’Immunologia, Hospital Clínic de Barcelona, Barcelona, Spain 3Department of Apoptosis in Hematopoietic Stem Cells, Helmholtz Center Munich, German Center for Environmental Health (HMGU), Munich, Germany 4 Department of Pediatrics, Dr von Hauner Children’s Hospital, LMU, Munich, Germany 5 Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain 6 Centro de Investigación Biomédica en Red-Oncología (CIBERONC), Instituto de Salud Carlos III, Madrid, Spain 7 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Correspondence Dr. Pablo Menéndez and Diego Sánchez- Abstract Martínez, Department of Biomedicine, CD19-directed chimeric antigen receptors (CAR) T cells induce impressive rates Josep Carreras Leukemia Research Insti- tute, School of Medicine, University of of complete response in advanced B-cell malignancies, specially in B-cell acute Barcelona, Barcelona, Spain. lymphoblastic leukemia (B-ALL). However, CAR T-cell-treated patients eventu- Email: pmenendez@carrerasresearch.org; ally progress due to poor CAR T-cell persistence and/or disease relapse. The bone dsanchez@carrerasresearch.org marrow (BM) is the primary location for acute leukemia. The rapid/efficient Funding information colonization of the BM by systemically infused CD19-CAR T cells might European Research Council, Grant/Award enhance CAR T-cell activity and persistence, thus, offering clinical benefits. Numbers: CoG-2014–646903, PoC-2018- 811220; Spanish Ministry of Economy and Circulating cells traffic to BM upon binding of tetrasaccharide sialyl-Lewis X Competitiveness, Grant/Award Numbers: (sLeX)-decorated E-selectin ligands (sialofucosylated) to the E-selectin receptor MINECO, SAF2016-80481R, SAF2019- 108160R; Fundacion Uno entre Cienmil; expressed in the vascular endothelium. sLeX-installation in E-selectin ligands is “la Caixa” Foundation, Grant/Award achieved through an ex vivo fucosylation reaction. Here, we sought to charac- Number: LCF/PR/HR19/52160011; Spanish terize the basal and cell-autonomous display of sLeX in CAR T-cells activated Association against cancer (AECC, Semilla 2019) using different cytokines, and to assess whether exofucosylation of E-selectin ligands improves CD19-CAR T-cell activity and BM homing. We report that cell- autonomous sialofucosylation (sLeX display) steadily increases in culture- and in vivo-expanded CAR T cells, and that, the cytokines used during T-cell acti- vation influence both the degree of such endogenous sialofucosylation and the CD19-CAR T-cell efficacy and persistence in vivo. However, glycoengineered This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics Clin. Transl. Med. 2021;11:e280. wileyonlinelibrary.com/journal/ctm2 1 of 12 https://doi.org/10.1002/ctm2.280 2 of 12 SÁNCHEZ-MARTÍNEZ et al. enforced sialofucosylation of E-selectin ligands was dispensable for CD19-CAR T-cell activity and BM homing in multiple xenograft models regardless the cytokines employed for T-cell expansion, thus, representing a dispensable strat- egy for CD19-CAR T-cell therapy. KEYWORDS BM homing, CAR T-cells, E-selectin ligands, exofucosylation 1 INTRODUCTION ing activity to E-selectin receptor is specifically exerted by the sialofucosylated E-selectin ligands CLA, CD43E, Immunotherapy has revolutionized cancer treatment. and HCELL resulting from the sLeX instalment (α-2,3- Adoptive cell immunotherapy using T cells genetically sialic acid and α-1,3-fucose binding determinants on N- redirected to a tumor-specific antigen by chimeric anti- glycans) in the native E-selectin ligands PSGL1, CD43, gen receptors (CAR) has induced impressive rates of com- and CD44, respectively.13,14 Of note, native E-selectin lig- plete response in advanced B-cell malignancies, especially ands can be converted into sLeX-displaying (sialofucosy- in B-cell acute lymphoblastic leukemia (B- ALL).1,2 Unfor- lated) E-selectin ligands through a straightforward glycan tunately, however, CAR T-cell-treated patients eventu- engineering approach involving minimal ex vivo cellular ally progress due to either poor CAR T-cell persistence manipulation based on a α-1,3-fucosyltransferase enzy- and/or disease relapse. Common limitations associated to matic reaction and guanosine diphosphate-fucose (GDP- CD19-CAR T-cell treatment, but extendable to other CARs fucose) substrate.15–17 Such exofucosylation reaction was are: (i) failure to achieve complete remission, (ii) relapse previously shown to endow BM homing abilities to with potential antigen loss, (iii) cytokine release syndrome hematopoietic stem/progenitors cells (HSPCs), mesenchy- (CRS) and related toxicities, and (iv) existence of multi- mal stem/stromal cells (MSCs), and immune cells.15,18,19 treated patients not eligible for CAR T-cell therapy due to Here, using CD19-CAR T cells as a working model, low counts of T cells.3–5 we sought (i) to characterize the basal/cell-autonomous In the clinical practice, adoptive cell therapies are display of sLeX in CAR T-cells activated with either IL- systemically infused via the bloodstream. However, the 2 or IL-7/IL-15, and (ii) to assess whether exofucosyla- bone marrow (BM) is the primary location for acute tion of E-selectin ligands to enforce sLeX display improves leukemia initiation and maintenance.6–8 Anatomically, the CD19-CAR T-cell activity and BM homing. Our results BM microenvironment confers cellular interactions and revealed that cell-autonomous sialofucosylation steadily signals promoting leukemia initiation, maintenance and augments in culture- and in vivo-expanded CAR T cells, progression, as well as drug resistance of leukemic cells.9 and that the type of cytokines used during T-cell activa- Current challenges associated to CAR T-cell treatment in tion influences both the cell surface display of sLeX in acute leukemia patients might be partially overcome by CD19-CAR T cells and the CD19-CAR T-cell efficacy and a rapid and effective CAR T-cell redirection to the BM. persistence in vivo. However, enforced sLeX display in E- In fact, efficient seeding of systematically infused CAR selectin ligands by exofucosylation was dispensable for T cells in the leukemic BM might enhance CAR T-cell both CD19-CAR T-cell activity and BM homing, regardless activity and persistence, eventually providing key clinical the cytokines employed for T-cell expansion. Collectively, benefits associated to the potential reduction of the CAR glycoengineered sLeX display in CAR T-cells systemically T-cell dose infused, namely less procedure-related toxici- administered is a dispensable strategy for improved CAR ties, lower production costs, and broader patient’s inclu- T-cell function. sion criteria. Although several mechanisms regulate the homing of circulating cells to the BM,10–12 the ability of circulating 2 RESULTS cells to traffic to the BM initially relies on their robust adherence to the E-selectin receptor (CD62E) displayed 2.1 Cell-autonomous and in the vascular endothelium (VE). Adhesive interactions exofucosylation-enforced expression of between the E-selectin receptor and its cognate ligand, sLeX in CD19-CAR T cells tetrasaccharide sialyl-Lewis X (sLeX), displayed on circu- lating cells dictate adherence of circulating cells to the T cells from peripheral blood (PB) of healthy donors (n = 5) VE, the first step of such biological process.8 Cell bind- were activated using CD3/CD28 plus either IL-2 or IL-7/ SÁNCHEZ-MARTÍNEZ et al. 3 of 12 IL-15 and transduced on day 2 with CD19-CAR-expressing Regardless the cytokines used for T-cell activation, exo- lentivectors (Figure 1A). The levels of sialofucosylation fucosylation rendered HECA452 expression in 100% of (sLeX display in cell surface) were analyzed by FACS on the FTVII-treated CD19-CAR T cells while not affecting CAR T cells over the 9-day activation/expansion period the expression of the VLA-4, the putative VCAM-1 ligand, using the HECA452 MoAb, which recognizes sLeX.20 confirming the specificity of the FTVII treatment (Fig- Basal expression of sLeX (HECA452+) was found in ure 2C). BT- and FTVII-treated CD19 CAR T cells (upper approximately 31 ± 5% of T cells at day 0 (Figure 1B). chamber) showed identical migratory capacity toward tar- Interestingly, in vitro T-cell activation/expansion led to a get cells (bottom chamber) through either nonstimulated cell-autonomous gradual increase in sLeX-expressing CAR or TNF-α-stimulated HUVEC monolayers (Figure 2D), T cells (Figure 1B). Of note, IL-7/IL-15 activation con- which translated into identical cytotoxicity of target cells sistently rendered higher frequency of sialofucosylated by the migrating CAR T cells, in 24 h assays (Figure 2E). (HECA452+) CAR T cells than IL-2-based activation Taken together, enforced exofucosylation of CD19-CAR T (75 ± 7% vs 50 ± 5% at day 9; Figure 1B), in a T-cell prolifer- cells enhances neither cytotoxicity nor homing in vitro. ation/expansion independent manner (Figure 1C). Using a well-established FTVII-based exofucosylation reaction17 (Figure 1D), 100% of culture-expanded CAR T cells became 2.3 Exofucosylation enhances neither HECA452+ within 48 h, regardless the cytokines used homing to BM/spleen nor during T-cell activation (Figure 1E). Western Blot (WB) activity/persistence of CAR T cells in vivo analysis using E-selectin-Ig immunoprecipitates clearly identified CD43 (CD43E), and partially PSGL1 (CLA), as We next assessed whether enforcing sLeX display by exofu- the E-selectin ligands carrying sLeX in exofucosylated cosylation promotes rapid migration of CD19 CAR T cells CAR T cells (Figure 1F). We next analyzed the phenotype of to BM and spleen. A sum of 3 × 106 BT- or FTVII-treated the expanded T cells using a CCR7 and CD45RA staining, CD19-CAR T cells were i.v. infused in NSG mice previously and found that neither the cytokines used nor the exofuco- intra-BM transplanted with CD19+ target cells, and the sylation reaction affected the T-cell phenotype (TN, TCM, ability of CD19-CAR T cells to colonize the BM and spleen TEFF/EM, TEMRA) upon 9 days of expansion (Figure 1G). was analyzed as early as 24 and 72 h after (Supporting infor- Collectively, although the cytokines used for T-cell activa- mation Figure S1A). In line with the in vitro data, similar tion influence the level of sLeX display in CAR T cells, numbers of BT- and FTVII-treated CAR T cells were found cell-autonomous sialofucosylation gradually increases in in PB and BM 24 and 72 h (Figure S1B) after CAR T-cell culture-expanded CAR T cells, with up to 80% of CAR T infusion, regardless the cytokines used during T-cell stim- cells being endogenously fucosylated at the end of the in ulation. These data suggest that, at least as a “stand-alone” vitro expansion. strategy, exofucosylation of CAR T cells does not speed-up CD19-CAR T-cell colonization of BM. We next investigated whether exofucosylation endows 2.2 Exofucosylation enhances neither CD19-CAR T cells with an enhanced cytotoxic activity or cytotoxic activity nor homing of CAR T prolonged persistence. Three in vivo models using differ- cells in vitro entially aggressive targets cells were employed (Figures 3 and 4). A sum of 3 × 106 of IL-2-expanded BT- or FTVII- We first prompted to analyze in vitro the cytotoxic activity treated CD19-CAR T cells from different donors were i.v. of exofucosylated CD19-CAR T cells. The cytotoxicity and infused in NSG mice 3, 7, or 14 days after transplantation of specificity of both BT- (control) and FTVII-treated (sialo- Luc-expressing NALM6 (Figure 3A), SEM (Figure 3B), or fucosylated) CD19-CAR T cells were identical in in vitro B-ALL PDX (n = 2, Figure 4A), respectively. BT- or FTVII- assays against CD19+ (NALM6, SEM) and CD19- (Jurkat) treated CD19-CAR T cells were equally effective in con- cell lines at multiple effector:target ratios (Figure 2A). We trolling the leukemia overtime, regardless the target cells next investigated the potential of exofucosylated CD19- used (Figures 3C, D and 4B). FACS analysis of residual CAR T cells to migrate through TNF-α-stimulated Human target cells at sacrifice revealed identical cytotoxic activ- Umbilical Vein Endothelial Cells (HUVEC) monolayers ity of both BT- or FTVII-treated CD19-CAR T cells in con- using standard in vitro transwell migration assays (Fig- trolling NALM6 (Figure 3E), SEM (Figure 3F), and PDX ure 2B). As expected, the expression of both E-selectin B-ALL (Figure 4C) cells in PB, BM, and spleen. In addi- receptor and VCAM-1, a key vascular cell adhesion tion, endpoint analysis of CAR T cells revealed no per- molecule, was upregulated in HUVEC cells upon TNF-α sistence advantage of FTVII-treated effector CAR T cells stimulation, thus, mimicking an activated microvas- in PB, BM, and spleen (Figures 3G, H and 4D). We then culature environment21,22 (Figure 2B, right panels). infused limiting doses of BT- and FTVII-treated CAR T 4 of 12 SÁNCHEZ-MARTÍNEZ et al. F I G U R E 1 Cell-autonomous and enforced expression of sLeX in CAR T cells. (A) Experimental design and timing of ex vivo CAR T- cell activation, transduction and exofucosylation. (B) Basal expression and in vitro regulation of sLeX (detected by HECA452 MoAb) at the indicated time points in IL-2- or IL-7/IL-15-activated T cells (n = 5 independent donors). Horizontal dotted line depicts the basal expression of HECA452 before T- cell activation. Right panels, representative FACS analysis of sLeX (HECA452) at the end of the activation protocol. Vertical dotted lines represent the isotype staining. (C) Very similar T-cell expansion at the end (day 9) of the in vitro culture protocol under SÁNCHEZ-MARTÍNEZ et al. 5 of 12 cells (in the SEM model) to more accurately assess whether subverted tumor vasculature, impediments of CAR T-cell FTVII-treatment may provide an improved in vivo cyto- trafficking, and immunesuppressive microenvironment.26 toxic activity of CAR T cells when administered in limited Similarly, the primary location for acute leukemogenesis is numbers, and found that exofucosylation did not endow the BM, and the BM microenvironment provides leukemic CAR T cells with an improved antileukemia effects regard- cells with cellular interactions and signals promoting less the cell dose infused (Supporting information Figure leukemia initiation, progression, and chemoresistance.6–9 S2). We finally measured the frequency of sialofucosylated However, CAR T cells in patients suffering from acute (HECA452+) CAR T cells in both BM and PB at sacrifice of leukemias are systemically infused via the bloodstream. the B-ALL PDX models, and found identical levels (∼80%) Practically all cellular therapies systemically admin- of sLeX display in both BT- and FTVII-treated CAR T cells istered to treat hematological malignancies such as (Figure 4E), further indicating cell-autonomous sialofuco- transplantation of unmodified or gene therapy-modified sylation of in vivo-expanded CAR T cells. HSPCs,27 or infusion of donor unmodified immune cells Of note, the results from these three in vivo models rely on efficient seeding in the leukemic BM.28 Similarly, were fully reproduced with IL-7/IL-15-activated/expanded cell therapy based on MSCs for graft-versus-host disease or CAR T cells (Supporting information Figure S3), further inflammatory conditions also rely on successful MSC traf- validating that glycoengineered sLeX display in CAR T ficking/homing to the damaged tissue.29,30 Here, we have cells is dispensable for CAR T-cell activity and persis- hypothesized that CAR T-cell immunotherapies (CD19- tence, regardless the cytokines used during T-cell stim- CAR as a working model) in acute leukemia patients may ulation. However, regardless enforced exofucosylation of also benefit from a rapid and effective redirection of effec- CAR T cells, IL-2-expanded CD19-CAR T-cells displayed tor cells to BM. Efficient seeding of infused CAR T cells a better control of the disease coupled to a higher T-cell in the leukemic BM might enhance their activity and per- persistence than IL-7/IL-15-expanded CD19 CAR T cells sistence, eventually providing many clinical benefits asso- in all the in-vivo leukemia models used (NALM6, SEM, ciated to the potential reduction in the CAR T-cell dose and PDXs, Figures 3 and 4 versus Supporting information to be infused, namely less CRS, lower production costs, Figure S3). and broader patientťs inclusion criteria. Previous stud- ies from several laboratories have suggested that enforced expression ex vivo of E-selectin ligands (exofucosylation) 3 DISCUSSION leads to transendothelial migration of systemically admin- istered HSPCs, MSCs, and T cells at E-selectin-expressing CAR T-cell therapy has been acclaimed as a revolution endothelial beds.16,19,31–33 Here, we have addressed the role in cancer treatment following the impressive results in of cell-autonomous and enforced sialofucosylation (sLeX hematological B-cell malignancies, especially in refrac- display) in E-selectin ligands in the cytotoxic activity and tory/relapse B-ALL. However, despite the impressive homing ability of systemically administered CD19 CAR T response rates, CD19-directed adoptive cell immunother- cells. Taking advantage of state-of-the-art in vitro assays apy is on its infancy, and unfortunately, a large proportion as well as short- and long-term in vivo xenograft models of CD19-CAR T-cell-treated patients eventually progress using several B-ALL cell lines and PDXs, our FACS and bio- due to either poor CAR T-cell persistence and/or disease chemical data revealed that cell-autonomous sialofucosy- relapse.23,24 Indeed, many studies in the coming years lation steadily increases in culture- and in vivo-expanded are expected to seed light into key molecular and cel- CAR T cells. In contrast, a study by Mondal et al. has lular immunological mechanisms underlying CAR T-cell recently shown that in vitro-expanded CAR T cells do not biology.25 Furthermore, CAR T cells for solid tumors are exhibit sLeX expression/E-selectin binding capacity.34 One lagging behind in part because the need to circumvent may attribute such differences to the use of different scFvs, the physical barriers of the tumor architecture such as T-cell activation conditions, biological differences of the both IL-2- and IL-7/IL-15-activation conditions. (D) Cartoon of the ex vivo exofucosylation reaction using fucosyltransferase-VII (FTVII) and GDP-fucose. Exofucosylation of PSGL-1, CD43, and CD44 generates the corresponding glycomodified E-selectin ligands CLA, CD43E, and HCELL, respectively. (E) Representative FACS analysis of sLeX (HECA452) expression in IL-2- or IL-7/IL-15-activated T cells after buffer-(BT) or FTVII-treatment (n = 5). (F) Western blot analysis of E-selectin reactive glycoproteins in naïve T cells, KG1a cells (positive control), and BT or FTVII-treated CAR T cells. E-selectin-Ig immunoprecipitated (IP) glycoproteins from BT or FTVII-treated CAR T cells were blotted against (PSGL- 1, CD43, and CD44). Representative image of n = 3 independent donors. (G) Top panels, a representative FACS plot depicting how TN, TCM, TEFF/EM, TEMRA T-cell subsets were identified using a CCR7 and CD45RA staining. Bottom panels, relative proportion of TN, TCM, TEFF/EM, TEMRA cell subsets after IL-2- versus IL-7/IL-15-based T-cell activation or after exofucosylation (FTVII- versus buffer treatment) 6 of 12 SÁNCHEZ-MARTÍNEZ et al. F I G U R E 2 Exofucosylation enhances neither in vitro cytotoxicity nor homing of CAR T-cells on TNF-α-activated HUVEC cells. (A) IL-2- and IL-7/IL-15-activated CAR T cells were BT- or FTVII-treated before in vitro exposure (24 h) to NALM6, SEM, and Jurkat at the indicated Effector:Target ratio. The alive (7-AAD-) eFluor 670+ target cells were determined by FACS (n = 3, independent donors). (B) Cartoon depict- ing the transwell assay using HUVEC monolayers for analysis of CAR T-cell (upper) migration toward target cells (bottom). HUVECs were stimulated with TNF-α-stimulated for 4 h for expression of VCAM-1 and E-selectin expression (right panels). (C) Representative FACS of the expression of the VCAM-1 ligand VLA-4 (left panels) and the exofucosylated E-selectin ligands (sLeX/HECA452-expressing, right panels) in IL-2- and IL-7/IL-15-activated CAR T cells after BT- or FTVII treatment (n = 3). (D,E) Absolute number of migrating BT- or FTVII-treated CAR T cells (D) and alive (7ADD-) target cells (E) quantified in the bottom chamber after 24 h (n = 6 donors in three independent experiments). CAR T cells were activated either with IL-2 or IL-7/IL-15. The assays were performed using unstimulated or TNF-α-stimulated HUVECs. *P < .05, **P < .01, ***P < .001 SÁNCHEZ-MARTÍNEZ et al. 7 of 12 F I G U R E 3 Exofucosylation is dispensable for CAR T-cell activity and BM homing in both NALM6 and SEM in vivo models. (A,B) Schematic of CAR T-cell activity and homing using NALM6 (A) or SEM (B) as target cells. NSG mice (n = 6 per group) intra-BM transplanted with 1 × 105 Luc-expressing NALM6 cells were i.v. infused 72 h later with 3 × 106 BT- or FTVII-treated CAR T cells. NALM6/SEM engraftment was followed weekly by bioluminescence. Target cell and T-cell engraftment was FACS analyzed in PB, BM, and spleen at sacrifice (for NALM6, day +12 for Mock T cells and day +25 for CAR T-cells; SEM, day +34). (C,D) IVIS imaging of NALM6 (C) and SEM (D) tumor burden monitored by BLI at the indicated time points. Bottom panels, total radiance quantification (p/s/cm2/sr) at the indicated time points. †: sacrifice. (E,F) NALM6 (E) and SEM (F) engraftment in PB, BM, and spleen at sacrifice in control mice (Mock T-cells), BT- and FTVII- treated CAR T-cells. (G,H) Persisting CAR T cells in PB, BM, and spleen at sacrifice in control mice, BT-, and FTVII-treated CAR T cells in the NALM6 (G) and SEM (H) model 8 of 12 SÁNCHEZ-MARTÍNEZ et al. F I G U R E 4 Exofucosylation is dispensable for CAR T-cell activity and BM homing in B-ALL PDX models. (A) Schematic of CAR T-cell activity and homing experiment using two independent B-ALL PDX (n = 2). NSG mice (n = 6 per group) were i.v. transplanted with 5 × 105 Luc-expressing PDX cells and 3 × 106 BT- or FTVII-treated 3 × 106 CAR T-cells were i.v. infused 14 days later, after engraftment was determined by FACS. Mice were distributed equally in each group. PDX engraftment was followed weekly by BLI. Tumor burden and T-cell engraftment was analyzed in PB and BM at sacrifice (day +33). (B) IVIS imaging of tumor burden monitored by BLI at the indicated time points. Right panel, total radiance quantification (p/s/cm2/sr) at the indicated time points. †: sacrifice. (C) Tumor burden in PB and BM at sacrifice in control (Mock T-cells) mice, BT- and FTVII-treated CAR T cells. (D) Persisting CAR T cells in PB and BM at sacrifice in control mice, BT-, and FTVII-treated CAR T-cells. (E) Expression of sLeX (HECA452) in BT- and FTVII-treated CAR T cells circulating in PB and present in BM at sacrifice SÁNCHEZ-MARTÍNEZ et al. 9 of 12 donor T-cells employed (HLA haplotypes, age, comorbil- CD19-CAR T cells in all the in-vivo leukemia models ities), etc. We have systematically compared side-by-side employed in the present study (NALM6, SEM, and PDXs), IL-2- versus IL-7/IL-15-based T-cell activation conditions, suggesting that adequate T-cell expansion protocols may concluding that the cytokines used for T-cell activation benefit the manufacturing and clinical outcome of CAR T influence the degree of cell-autonomous sLeX expression cells. Collectively, our results support that the cytokines in CAR T cells. However, regardless of the cytokines used used during T-cell activation influence both the degree for T-cell activation, cell-autonomous sLeX expression/E- of cell-autonomous sialofucosylation and the CD19-CAR selectin binding capacity gradually increased in culture- T-cell efficacy and persistence in vivo. However, at least and in vivo-expanded CAR T cells. Moreover, identical as a “stand-alone” strategy, glycoengineered exosialofu- levels of sLeX expression/sialofucosylation were observed cosylation of E-selectin ligands seems dispensable for in in vitro-expanded CAR+ and CAR-T cells, suggesting CD19-CAR T-cell activity and BM homing in multiple a CAR-independent cell-autonomous sialofucosylation of xenograft models regardless of cytokines employed for activated culture-expanded T cells (data not shown). T-cell expansion. Which and how alternative cellular Our results using multiple in vitro and in vivo xenograft and molecular mechanisms regulate the migration of models revealed that further enforced sLeX-installation in circulating CAR T-cells to BM needs to be explored in E-selectin ligands improves neither the cytotoxic activity further studies. nor BM/spleen homing of vascularly administered CD19- CAR T cells, regardless of the cytokines used for T-cell activation. Of note, the T-cell phenotype was not altered 4 MATERIALS AND METHODS by either the cytokines used for T-cell expansion or the exofucosylation reaction. This is in line with the reported 4.1 CD19-CAR vector, lentiviral cell-autonomous steady sialofucosylation of in vitro production, T-cell transduction, activation culture-expanded CAR T cells prior to in vivo infusion. and expansion Furthermore, the frequency of HECA452+ CAR T cells was found very similar in xenografts infused with either Our clinically validated pCCL lentiviral second-generation BT- or FTVII-treated CAR T cells, suggesting that cell- CD19CAR backbone containing a human CD8 trans- autonomous sialofucosylation of T cells in vivo seems membrane (TM) domain, human 4-1BB and CD3z sufficient for proper in vivo effector function. A major endodomains, and a T2A-GFP cassette has been reported difference between our experimental design and that by elsewhere.36,37 CAR-expressing viral particles pseudo- Mondal et al. is that, in our study, CAR T cells were infused typed with VSV-G were generated in 293T cells using in NSG mice previously transplanted with CD19+ target standard polyethylenimine transfection protocols, and cells, thus, making our in vivo model more informative. were concentrated by ultracentrifugation as described It should be noted that CAR T cells are not expected elsewhere.38 Viral titers were consistently in the range of to migrate or persist in BM or spleen in the absence of 108 TU/mL. Peripheral blood mononuclear cells (PBMCs) target antigen. Therefore, our xenograft models permit were isolated from buffy coats from healthy volunteers a physiologically more relevant in vivo assessment of by using Ficoll–Hypaque gradient centrifugation. Buffy (i) the trafficking ability of the infused exofucosylated coats were obtained from the Barcelona Blood and CAR T cells in the presence of target antigen-expressing Tissue Bank upon institutional review board approval leukemic niches, and (ii) the cytotoxic activity of exofuco- (HCB/2018/0030). T cells were plate-bound activated with sylated CAR T cells. In a different adoptive immunother- anti-CD3 and anti-CD28 antibodies for 2 days and then apy context, tumor infiltrated lymphocytes (TILs) have transduced with CAR-expressing lentivirus (multiplicity been recently reported to display an increased in vivo of infection = 10) in the presence of either interleukin- but also in vitro cytotoxic activity upon exofucosylation, 2 (IL-2, 50 UI/mL, Mitenyi Biotec) or IL-7 and IL-15 suggesting that the enhanced sLeX expression seemed (10 ng/mL, Mitenyi Biotec).38 Proper CAR expression, important for the target cell recognition.35 Furthermore, T-cell activation, and expansion was confirmed at the end the mechanisms for activation/expansion and target cell of the activation period, as previously described.38 recognition of TILs clearly differ from those from CAR T cells, further explaining such experimental discrepan- cies between the distinct effector cells used for adoptive 4.2 Exofucosylation reaction cell immunotherapies. Of note, regardless enforced exo- fucosylation of CAR T cells, IL-2-expanded CD19-CAR CD19-CAR T cells expanded for 9 days either with IL-2- T cells showed a better control of the disease coupled or IL-7/IL-15 were treated on Hanks’ Balanced Salt solu- to a higher T-cell persistence than IL7-/IL-15-expanded tion (0.1% human serum albumin and 10 mM HEPES) 10 of 12 SÁNCHEZ-MARTÍNEZ et al. with GDP-fucose (Biosynth Carbosynth, Compton, UK) 4.4 HUVEC transwell assays and FTVII (RD Systems). One million cells were incu- bated in 20 μL of buffer containing 1 mM of GDP-fucose HUVEC were maintained in EGM-2 Endothelial Cell and 70 μg/mL of purified FTVII enzyme at 37◦ C for 1 h Growth Medium-2 BulletKit (Lonza, Cultek SLU), as pre- as previously detailed.17 Control cells were incubated in viously described.40 Early passage HUVECs were plated on the same solution but without FTVII/GDP-fucose (buffer- 24-well Transwell plates (5 μm polycarbonate membrane, treated [BT] cells). After the enzymatic reaction, cells were 6.5 mm insert), and stimulated with 40 ng/mL of TNF-α always washed twice with PBS before downstream experi- (R&D) for 4 h at 37◦ C to activate cell surface expression of ments. E-selectin and VCAM-1.17 A sum of 2 × 105 of each NALM6 cells and BT- or FTVII-treated CAR T cells were seeded in the bottom and upper chamber, respectively. Twenty-four 4.3 E-selectin-Ig immunoprecipitation hours later, the absolute number of alive (7AAD-) NALM6 and CAR T cells present in the bottom chamber were quan- CAR T-cell lysates were prepared using lysis buffer con- tified using Trucount tubes (BD Biosciences).38 taining 150 mM NaCl, 50 mM Tris-HCI (pH 7.4), 2% Non- idet P-40, 2 mM CaCl2, and protease and phosphatase inhibitor cocktails (Roche). When indicated, 5 mM EDTA 4.5 In vivo NALM6, SEM, and B-ALL was added to the lysis buffer as negative control condi- patient-derived xenograft (PDX) models tion. A sum of 5 × 106 cells were pelleted per condition, washed with PBS, and lysed in 500 μL of lysis buffer. Six- to twelve-week-old nonobese diabetic NOD.Cg- Cell lysates were incubated on ice for 15 min and cen- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice (Jackson Laboratory) trifuged at 12 000 g for 10 min. Cell lysates were then pre- were bred and housed under pathogen-free conditions in cleared overnight using protein G-agarose beads (Roche) the animal facility of the Barcelona Biomedical Research and incubated for 2 h at 4◦ C with 3 μg of murine E-selectin- Park (PRBB). All in vivo experimental procedures were human Fc chimera (“E-Ig,” R&D Systems), as described.17 performed in compliance with the institutional animal Agarose beads were then washed twice with lysis buffer, care committee of the PRBB (DAAM7393). Briefly, irra- and immunoprecipitated glycoproteins were collected by diated (2 Gy) mice (5-6/condition) were intra-BM or i.v. boiling the beads in the presence of 2-mercaptoethanol transplanted with Luc/GFP-expressing SEM (1 × 105 ), in Laemmli loading buffer. For western blot (WB) anal- NALM6 (1 × 105 ), or B-ALL PDX (5 × 105 , PDX-50/PDX- ysis, immunoprecipitates were resolved on a 7.5% SDS- 265) cells and then i.v. infused with 3 × 106 BT- or PAGE gel (Bio-Rad Laboratories) and then transferred onto FTVII-treated CD19-CAR T cells when engraftment was a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). detectable. An in vivo experiment was performed with The membrane was then blocked with blocking reagent SEM cells where decreasing doses (2 × 106 , 1 × 106 , (Chemiluminescence Western Blotting Kit, Roche), and 0.5 × 106 , 0.2 × 106 ) of BT- or FTVII-treated CD19-CAR T incubated with monoclonal antibodies (MoAb) against cells were infused. Both IL-2- and IL-7/IL-15-based CAR PSGL1 (clone KPL1, BD), CD43 (clone IG10, BD), and T-cell activation protocols were used. Tumor burden was CD44 (clone 2C5, R&D). Protein bands were detected by monitored weekly by bioluminescence (BLI) using the chemiluminescence using Lumi-Light substrate (Chemi- Xenogen IVIS 50 Imaging System (Perkin Elmer).38 Tumor luminescence Western Blotting Kit). burden (HLA-ABC+CD45+CD19+CD22+CD3-) and CAR T-cell persistence (HLA-ABC+CD45+CD3+GFP+) were also quantified by FACS at sacrifice in BM, PB, and spleen. 4.3.1 In vitro cytotoxicity assays Luciferase (Luc)/GFP-expressing-NALM6 cells were 4.6 FACS analysis kindly provided by Prof. RJ Brentjens (MSKCC, NY). SEM were generated by retroviral transduction and GFP-based Cell staining and FACS analysis were performed as exten- FACS-selection.39 Jurkat were purchased from DSMZ. sively described38 using a FACSCanto-II flow cytome- Target cells were labeled with 3 μM eFluor670 (eBio- ter equipped with FACSDiva software (BDBioscience). science) and incubated with BT- and CD19-CAR T cells at Briefly, 0.5 × 105 total cells recovered from in vitro or in different Effector:Target (E:T) ratios. CAR T-cell-mediated vivo assays were stained in PBS + 2%FBS with the fol- cytotoxicity was determined by analyzing the residual lowing MoAb: HECA-452 (CLA)-BV421, CD62/E-Selectin- alive (7-AAD-) eFluor670+ target cells after 24 h CAR APC, CD49d/VLA-4-PE, VCAM-1-APC, HLA-ABC-PE, T-cell exposure. CD45-BV421, CD3-PerCP, CD45RA-AmCyan, CCR7-PE, SÁNCHEZ-MARTÍNEZ et al. 11 of 12 CD22-APC, and CD19-BV421. IgG1-APC, IgG1-PE, and Rat AVA I L A B I L I T Y O F D A T A A N D IgM-BV421 were used as isotype controls. 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