CD19 positive human Burkitt’s lymphoma cell lines Daudi, NALM-6 and Raji (American Type Culture Collection Company (USA)) and CD19 negative Non-Hodgkin’s Large Cell Lymphoma cell line Karpas-299 (Sigma Aldrich) were maintained in RPMI 1640 (EuroClone, Italy) supplemented with 10% heat-inactivated fetal bovine serum (EuroClone), 2 mM l-glutamine (GIBCO, USA), 25 IU/mL of penicillin, and 25 mg/mL of streptomycin (EuroClone), in a humidified atmosphere containing 5% CO2 at 37°C. All cell lines were authenticated by PCR-single-locus-technology (Promega, USA. PowerPlex 21 PCR) analysis in ‘BMR Genomics s.r.l.’ (Italy), and were periodically checked for mycoplasma (VenorGeM Advance, MB Minerva biolabs, UK) and surface markers expression.

Buffy coats (BC) from healthy donors (HDs), peripheral blood (PB) and bone marrow (BM) derived from children with Bcp-ALL were used to isolate unfractionated mononuclear cells using Lympholyte Cell Separation Media (Cedarlane, Canada). T-cells were activated with OKT3 (1 µg/mL, ThermoFisher Scientific, USA) and anti-CD28 (1 µg/mL, BD Biosciences, USA) monoclonal antibodies (mAb) with a combination of recombinant human interleukin-7 (rh-IL7, 10 ng/mL; Bio-Techne; USA) and (rh-IL15, 5 ng/mL; Bio-Techne; USA). NK-cells were generated from BC of HDs following a previously described method.7 Then, after 3/4 days, T- and NK-cells were transduced with retroviral supernatant, in 24-well plates precoated with recombinant human RetroNectin (Takara-Bio; Japan). T lymphocytes were expanded in the presence of cytokines, in TexMacs complete medium (Miltenyi, Germany) and replenished twice a week.

Four different retroviral CAR constructs were used to carry out the experiments: CAR construct carrying anti-human CD19-scFv from FMC63 clone in which VL and VH fragments were joined by a linker represented by three GSSSS repetitions (3xG4S, long linker, LL), in frame with CD8 stalk domain (short hinge, SH), CD8 transmembrane domain, 4.1bb and CD3ζ cytoplasmic domain (CAR.CD19 VL-3GS-VH-CD8-4.1bb.ζ, i.e., CAR.CD19_LL/SH); CAR construct carrying anti-human CD19-scFv from FMC63 clone in which VL and VH fragments were joined by a linker represented by three GSSSS repetitions (3xG4S, LL), in frame with 16aa sequence derived from human CD34 antigen (ΔCD34, long hinge, LH), CD8 stalk domain, CD8 transmembrane domain, 4.1bb and CD3ζ cytoplasmic domain (CAR.CD19 VL-3GS-VH-CD34-CD8-4.1bb.ζ, i.e., CAR.CD19_LL/LH); CAR construct carrying anti-human CD19-scFv from FMC63 clone in which VL and VH fragments were joined by a linker represented by one GSSSS repetition (G4S, short linker, SL), in frame with CD8 stalk domain (SH), CD8 transmembrane domain, 4.1bb and CD3ζ cytoplasmic domain (CAR.CD19 VL-1GS-VH-CD8-4.1bb.ζ, i.e., CAR.CD19_SL/SH); CAR construct carrying anti-human CD19-scFv from FMC63 clone in which VL and VH fragments were joined by a linker represented by one GSSSS repetition (G4S, SL), in frame with 16aa sequence derived from human CD34 antigen (ΔCD34, LH), CD8 stalk domain, CD8 transmembrane domain, 4.1bb and CD3ζ cytoplasmic domain (CAR.CD19 VL-1GS-VH-CD34-CD8-4.1bb.ζ, i.e., CAR.CD19_SL/LH). NALM-6 genetically modified with a lentiviral vector carrying CAR.CD19 already published,5 have been kindly provided by Prof. Ruella under a specific material transfer agreement.

T-cells from HDs or Bcp-ALL patients as well as B leukemic cell lines including DAUDI, RAJI and NALM-6 were genetically modified using the retroviral construct CAR.CD19_SL/LH or CAR.CD19_LL/SH. NALM-6 cells were also genetically modified with CAR.CD19_LL/LH or CAR.CD19_SL/SH whereas NK-cells from HDs were genetically modified using only the retroviral construct CAR.CD19_SL/LH. CAR+ B-cell lines were FACS sorted for CAR expression after transduction.

Fluorescence Activated Cell Sorter (FACS) analysis was performed to determine cell surface antigen expression; mAb for CD45, CD3, CD19, CD22, CD10, CD34 (all from Becton Dickinson, USA) were combined with different fluorescence according to needs. CAR.CD19 expression was detected using a mAb directed to hCD34 epitope (anti-CD34 QBend-10 PE from Bio-Techne, USA), or CD19 CAR Detection Reagent (Biotin; Miltenyi, Germany). Flow cytometry analysis was performed using a BD LSRFortessa X-20 cytometer (BD Biosciences, USA) and analyzed by FACSDiva software (BD Biosciences, USA). FACS sorting of CAR-transduced tumor cell lines was performed on a FACSAria (BD Biosciences, USA). DPs were also characterized by either an 11-color or a 16-color combination of antibodies plus CD19-FITC human protein (anti-CAR TCD19, Cytognos SL, Salamanca, Spain), using the EuroFlow standard operating procedures (SOP) for staining of surface markers only, available at www.EuroFlow.org.8 Antibody combinations used were both based on a backbone consisting of the EuroFlow Bcp-ALL MRD tubes previously described for high-sensitive MRD measurements in Bcp-ALL by flow cytometry9–11 to which the anti-CD3 and both anti-CD22 and anti-HLADR antibody reagents were added for staining of transfected and non-transfected (NT) T-cells and specific gating of CD19-negative B-cell precursors and blasts, respectively. Finally, the anti-CD34 Qbend10 clone and the CD19-FITC human protein (Cytognos SL, Salamanca, Spain) were also added to the reagent staining mix for identification of transfected CAR.CD19 cells. Sample acquisition was performed immediately after sample preparation was completed, and >1.5×10⁶ cells (range: 1.6–7.5×10⁶ cells) were measured per sample using an LSRFortessa X-20 flow cytometer and the FACSDiva software or a 3-laser Aurora (Cytek Biosciences, California, USA) spectral flow cytometer equipped with the SpectroFlo software (Cytek Biosciences, USA). For instrument setup and data acquisition, the EuroFlow SOP for instrument setup and calibration available at www.euroflow.org was strictly followed.8 The Infinicyt software (Cytognos SL, Salamanca, Spain) was used for data analysis.

Cellular pellets for DNA and RNA extraction were obtained after repeated (three times) and prolonged (10 min at 800 rpm) cell washing in PBS (10 mL), to avoid nucleic acid carry-over from culture. Total RNA was purified with RNeasy Micro Kit (Qiagen, Maryland, USA) according to manufacturer’s instructions and reverse transcribed (SuperScript VILO cDNA Synthesis Kit, ThermoFisher Scientific, Italy) in cDNA. Validated assays were considered for TaqMan primers/probes specific for each mRNA analyzed (Hs_00174333 for hCD19, Hs_01060665 for hß-ACTIN, ThermoFisher Scientific). Total DNA was purified by QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. TaqMan primer/probes were designed for each specific IG or T-cell receptor (TR) clonal target by Primer Express software (Applied Biosystems, Italy). Sequences were reported in online supplemental table 1. Relative gene expression was calculated using the house-keeping gene ACT1N1 (Hs_02249516 ACT1N1, ThermoFisher Scientific, USA). qPCR were performed employing QuantStudio 12K Flex Real-Time PCR System (ThermoFisher Scientific, USA). For IG/TR PCR-minimal residual disease (MRD) of the DPs, each MRD value was calculated from the corresponding standard curve, and the results were normalized for values of the housekeeping Albumin gene. Quantitative range and sensitive range (SR), positive value, reproducibility of replicates, were interpreted following the Euro MRD guidelines, in order to assign the appropriate MRD value to each sample analyzed, and are reported in Supplemental Table 1 table 1. The reported value of MRD is referred to the value detected at diagnosis of each patients an arbitrary considered equal to 1. qPCR was performed by using the 7900 HT fast-Real Time-PCR System and ViiA7 system (ThermoFisher Scientific, USA) and TaqMan Gene Expression Master Mix (ThermoFisher Scientific, USA).

We selected all the class I human leukocyte alleles (HLA), as reported from the database allele frequency, found in more than 10% of the individuals from any of the following datasets: USA NMDP European Caucasian, USA NMDP Middle Eastern or North Coast of Africa, USA NMDP South Asian Indian, USA NMDP Southeast Asian, USA NMDP American Indian South or Central America, USA NMDP African, and USA NMDP Chinese. The HLA alleles selected are: A*01:01, A*02:01, A*03:01, A*11:01, A*23:01, A*24:02, A*33:03, B*07:02, B*08:01, B*40:01, B*46:01, and B*53:01.

We used the web server NetMHCpan 4.1 to predict HLA binders for all the alleles listed before, for any peptide in the junction regions of the CAR hinge domain plus/minus 12 residues, with a length between 8 and 12 amino acids. Only peptides that passed the following criteria were further analyzed: (1) The peptide must be predicted as a strong binder for at least one of the alleles; (2) The peptide must be encompassing at least two different regions; (3) Peptides that only have the N-terminal residues before the anchor position from one region, while all the rest is from another region, were discarded.

Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) female mice were provided by Charles River and maintained in the Plaisant animal facility in Castel Romano, Rome, Italy. All procedures were performed in accordance with the Guidelines for Animal Care and Use of the National Institutes of Health (Ethical committee for animal experimentation Prot. N 088/2016-PR). Tumor growth was monitored weekly by In Vivo Imaging System (IVIS).12 13 To test CAR T-cell activity on CAR+ leukemia cells, the NSG mouse model was intravenously engrafted with 0.25×10⁶ NALM-6 WT or NALM-6 CAR.CD19_SL/LH or NALM-6 CAR.CD19_LL/SH or DAUDI CAR.CD19_SL/LH cells genetically modified with firefly luciferase (FF-Luc). On day+3, mice were treated with 10×10⁶ CAR.CD19 T-cells14–18 or NT control T-cells. Tumor growth was monitored weekly by IVIS,12 13 after D-Luciferin (PerkinElmer, USA) intraperitoneal administration.

Unless otherwise noted, data are summarized as mean±SD. Student’s t-test (two sided) was used to determine statistically significant differences between samples, with a p<0.05 indicating a significant difference.

Mouse survival data were analyzed using Kaplan-Meier survival curves and the Fisher’s exact test was used to measure statistically significant differences. No valuable samples were excluded from the analyses. Animals were excluded only in the event of death after tumor implant, but before treatment. Neither randomization nor blinding was done during the in vivo study. However, mice were matched based on the tumor signal for control and treatment groups before infusion of control or specific CAR T-cells. To compare the growth of tumors over time, bioluminescence signal intensity was collected in a blind fashion. Bioluminescence signal intensity was log-transformed and, then, compared using a two-sample t-test. We estimated the sample size considering no significant variation within each group of data. We tried to reach a conclusion using a sample size as small as possible. We estimated the sample size to detect a difference in averages of 2 SD at the 0.05 level of significance with an 80% power. Graphic representations and statistical analysis were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, California, USA).

Unfractioned PB mononuclear cells isolated from patients at diagnosis had a mean of 45.05%±28.12% contaminating blasts (n=15; range: 5.5%–86.4%). Mononuclear cells derived from PB of the enrolled patients were transduced with a second-generation CAR.CD19 incorporating an SL between VL/VH and a CD34 16aa peptide in the hinge region (CAR.CD19_SL/_LH), according to the method detailed in figure 1A. At the end of production, T-cell products have shown a CAR transduction efficiency of 55.15%±16.54% (figure 1B shows a representative analysis, whereas figure 1C shows the average of 15 leukemia patients divided into two groups, based on the percentage of CD19+ cells in the starting material (SM), considering 45% as cut-off). We sought to evaluate whether leukemia blast contamination in the patients’ SM had any impact on the production of CAR T-cells, especially in terms of CAR transduction level in the DPs, as well as on the expansion rate of engineered CAR T-cells. We did not find any correlation between CAR T-cell percentage at the end of the manufacturing procedure and the level of CD19+ B cells contaminating the SM (figure 1C,D). By contrast, the number of T-cells was reduced in the SMs of patients with >45% of CD19+ contaminating blasts (day 0, online supplemental figure 1), although the difference with T-cells present in SM with <45% of CD19+ cells was not significant due to the small number of analyzed SMs. Irrespectively from the leukemia contamination of the SM, plated T-cells show comparable rate of expansion calculated from day 3 up to the end-production process (figure 1E), allowing to reach similar number of total T-cells in the DP (day 14; online supplemental figure 1). To evaluate patients' samples with an increased percentage of leukemia blast cells, we sought to generate CAR T-cells starting from BM samples of Bcp-ALL patients at diagnosis (n=10; online supplemental figure 2) in which the average of CD19+ cells in the BM SM was of 73.1%±17.80% (range: 40.5%–83.5%). Although the transduction level in BM-derived samples was significantly lower than in PB-samples (online supplemental figure 2A,B; 36.04±19.83% vs 55.15±16.54% CAR+ T-cells, respectively; p=0.03), also in this case we did not observe any difference in terms of total yield from PB or BM samples (online supplemental figure 2C).

We performed real-time quantitative PCR on CAR T-cell DPs (day+14, end of production) for the amplification of leukemia-specific IG rearrangements. We observed positivity of MRD in 7/14 tested CAR T-cell DPs, with a median MRD value of 6.01E-3±1.00E-2 (CAR, figure 2A). Moreover, we observed that leukemic blast contamination was present in 9/13 untransduced T-cell samples tested (NT, figure 2A) with a median value of 1.57E-3±1.86E-3. These results suggest that leukemic cells, although at minimal levels, may survive during CAR T-cell manufacturing for 14 days. In addition, to understand the dynamics of Bcp-ALL cell survival during manufacturing, we performed time-course experiments in which DPs were analyzed for MRD at a very early time point as of day+8 after activation, day+14 as standard procedures for CAR T-cell manufacturing, and day+30 as the most extended culture period for a CAR T-cell DPs (data from five different patients’ production). As shown in figure 2B, we observed an inverse correlation between MRD levels and time in culture, (p=0.02 considering MRD at day+8 vs day+14), reaching at the last time point (day+30) a value beyond the sensitivity level of the used method (online supplemental table 1 specify sensitivity range for each patient’s assay; SR 10⁻⁴-10⁻⁵). For those samples with enough available leftover material, we performed high-sensitivity EuroFlow flow cytometry analysis19 to better characterize Bcp-ALL cells contaminating the DPs. As shown in online supplemental figure 3, the applied flow cytometry test was sensitive enough to detect positive MRD in CAR T-cell DP at day+14. B-cell precursors contaminating CAR products resulted to be CD19 dim (online supplemental figure 3), although maintained other B-cell markers like CD10. On the contrary, the B-cells contaminating NT T-cells remained CD19+ and were significantly higher in number than those observed in CAR samples (online supplemental figure 3, CD19+ MRD of 1.5% vs 0.00036%, respectively). We then sought to verify whether B-cells were characterized by the presence of CAR transduction. As shown in figure 2C, for two samples analyzed by flow cytometry, we detected CAR+ leukemia cells showing double positivity for two different CAR-associated markers (anti-CD34, detecting the epitope on the hinge region of CAR.CD19_SL/LH as well as CD19 epitope recognized by CAR.CD19 scFv), although the number of detected CAR+ B-cells was at the limit of detection for ALL#12.

MRD data were also confirmed on BM-derived DPs, in which RT-qPCR showed MRD positivity in 6/6 CAR T-cell products (online supplemental figure 4A). For two BM-derived DPs, MRD positivity was also confirmed by the EuroFlow flow cytometry assay (online supplemental figure 4B). In BM-derived DPs, the sensitivity of the assay and the quality of frozen samples did not allow detection of CAR positive B-cell precursors. Moreover, in BM-derived CAR T-cell products, B-cell precursors contaminating the DPs were CD19 dim/negative, whereas untransduced T-cells have shown contaminating B-cells with high expression of CD19 (online supplemental figure 4B).

We sought to evaluate whether CAR.CD19 design in the linker and the hinge regions could have any impact on the CD19 masking when CAR.CD19 is coexpressed with CD19 on the same cellular membrane of tumor cells (in cis expression). In particular, we considered two CAR configurations with a relevant clinical interest. The first CAR is characterized by a standard configuration of LL between VL and VH derived from FMC63 clone, and a SH before the CD8 transmembrane domain represented by CD8 stalk (CAR.CD19_{LL/ SH}) (figure 3A, construct a).20 The second one, is represented by the CAR construct used in the current clinical trial NCT03373071 (at ‘Bambino Gesù Children Hospital’, Rome, Italy), in which the SL between VL and VH regions, is associated to the LH that includes ΔCD34, as trackable marker for CAR T-cells, placed before the CD8 stalk (CAR.CD19_{SL/ LH}) (figure 3A, construct b). After transduction with the two constructs, CAR+ NALM-6 maintained CD19 positivity at the mRNA level (online supplemental figure 5A). Nevertheless, the pattern of CD19 fluorescence detected by flow cytometry in CAR.CD19_LL/SH NALM-6 and CAR.CD19_SL/LH NALM-6 was reduced compared with CD19 expression in NALM-6 WT, tested either with the FMC63 (figure 3B) or HIB19 clone (figure 3C), confirming previously published data (online supplemental figure 6A shows the masking experiments on NALM-6 genetically modified with a lentiviral construct expressing a CAR.CD19_LL/SH, kindly provided by prof. Ruella).5 Notably, CAR.CD19_LL/SH NALM-6 cells showed lower CD19 Mean Fluorescent Intensity (MFI) than CAR.CD19_SL/LH NALM-6 (figure 3B,C), suggesting a lower CD19 masking in this last CAR+ cell line. The uncomplete CD19 masking was also confirmed in DAUDI and RAJI cell lines transduced with CAR.CD19_SL/LH (online supplemental figure 7, clone HIB19). To demonstrate which variable (linker or hinge) in CAR construct is responsible in regulating CD19 antigen masking in CAR+ leukemia cells, we proceeded considering other two different second-generation CAR.CD19 configurations in a retroviral platform (online supplemental figure 8A): one with LL and LH (CAR.CD19_LL/LH), (online supplemental figure 8A, construct a) and the second one, with SL and SH (CAR.CD19_SL/SH), (online supplemental figure 8A, construct b). Also in this case, masking flow cytometry analysis showed that CD19 MFI was significantly higher in NALM-6 expressing an SL (CAR.CD19_SL/SH NALM-6) than the same cell line expressing the CAR with the configuration of LL (CAR.CD19_LL/LH NALM-6; online supplemental figure 8B for FMC63 clone and online supplemental figure 8C for clone HIB19). Based on these observations, the linker length could be the factor driving a complete or uncomplete antigen masking when CAR.CD19 is coexpressed in cis with CD19 on CAR+ leukemia cells. These data were also corroborated by functional analysis. Indeed, very low level of CD19 expression on DAUDI, RAJI and NALM-6 CAR.CD19_SL/LH cells was sufficient to elicit CAR.CD19 T-cell response, (figure 4A–E). In particular, CAR.CD19 T-cells exert a significant leukemia control against NALM-6 WT, with no significant differences compared with the anti-leukemia activity observed against NALM-6 CAR.CD19_SL/LH (figure 4A) and NALM-6 CAR.CD19_SL/SH (online supplemental figure 8D). Notably, whereas CAR.CD19 T-cells were completely unable to recognize NALM-6 CAR.CD19_LL/SH in the lentiviral platform (online supplemental figure 6B) as reported in the previous publication,5 we observed some activity of CAR.CD19 T-cells against NALM-6 CAR.CD19_LL/SH, although to a lower extent compared with NALM-6 CAR.CD19_SL/LH (figure 4A) and NALM-6 CAR.CD19_SL/SH (online supplemental figure 8D). In line with these findings, we also observed that NALM-6 cells genetically modified with both CAR.CD19 with SL or LL were able to induce a significant amount of interferon-gamma (IFNγ) by CAR T-cells (online supplemental figures 9A and 8E), as well as to induce their proliferation (online supplemental figure 9B). Moreover, NALM-6 CAR.CD19_LL/SH induced a lower CAR.CD19 T-cell proliferation compared with WT and CAR.CD19_SL/LH NALM-6 cells (see P2 gates, online supplemental figure 9B).

Notably, whereas CAR.CD19_SL/LH is leading to the uncomplete CD19 antigen in cis masking, when expressed in T-cells it was able to exert a significant leukemia/lymphoma control. In particular, coculture assay was used to demonstrate the cytotoxic effect of CAR.CD19_SL/LH T-cells against NALM-6 (figure 4A,B), DAUDI (figure 4C) and RAJI (figure 4D) cell lines. As shown in figure 4B–D, CAR.CD19_SL/LH T-cells are able to significantly eliminate tumor cells from the culture even when used at low effector/target ratio. For the NALM-6 model, we have also compared the anti-lymphoma activity of CAR.CD19_SL/LH T-cells with that of the more standard CAR.CD19_LL/SH T-cells, not revealing substantial differences in terms of cytotoxicity (figure 4E, NALM-6 WT), IFNγ production (online supplemental figure 9A), or proliferation index after antigen stimulation (online supplemental figure 9B). This last assay was performed by stimulating CFSE loaded CAR T-cells with NALM-6 WT, and observing that irrespectively of the CAR construct, both CAR.CD19 T-cells were able to reach comparable level of proliferating cells (light gray histograms) compared with unstimulated cells (dark gray histograms). Moreover, since we have included in the LH CAR configuration the trackable marker CD34, we have also performed in silico analysis to predict its immunogenicity. In particular, we have studied the peptide sequences that are confidently foreseen to be presented by HLA molecules in the CAR region that include CD34 domain. ‘STNVSPAPR’ peptide is predicted as potentially immunogenic for CD34-including construct (online supplemental table 2), presented by the molecules HLA-A11:01 and HLA-A33:03. The peptide ‘GSELPTQGTF’ also matches the selection criteria, but in this case, its binding core is ‘ELPTQGTF’ and is entirely part of the CD34 epitope region, therefore unlikely to be highly immunogenic. For the CAR construct in which CD34 domain was not considered, the peptides ‘SVTVSSPAPR’ and its shorter version ‘SVTVSSPAPR’ are both predicted to be immunogenic, presented by the same alleles HLA-A11:01 and HLA-A33:03. In light of this finding, we predict that the inclusion of CD34 domain in the construct did not substantially affect the immunogenic profile of the CAR.

The ability of CAR.CD19 T-cells to target wild-type B-cell lymphoma beside CAR construct with an SL or a LL has been proved in vivo in a B-cell lymphoma NSG xenograft model. In particular, mice were infused systemically with NALM-6 genetically modified with FF-Luc to allow in vivo monitoring of the leukemia burden overtime. Tumor engraftment was analyzed by measuring the bioluminescence signal, and, on day+0, mice were treated with either CAR.CD19_SL/LH or CAR.CD19_LL/SH T-cells as well as with NT untransduced control T-cells (NT) derived from HDs (figure 5A-C). CAR.CD19_SL/LH and CAR.CD19_LL/SH T-cells were able to significantly control NALM-6 in vivo expansion, as clearly demonstrated by bioluminescence analysis. We then sought to evaluate whether NALM-6 CAR.CD19_SL/LH were also recognized by CAR T-cells in the in vivo setting. As shown in figure 5D-F, CAR.CD19_SL/LH T-cells were able to reduce significantly CAR+ NALM-6 cell in vivo expansion compared with NT. The same data were also confirmed in a less aggressive lymphoma model of DAUDI cell line (figure 6A). In this model, CAR.CD19_SL/LH T-cells were able to control and eliminate CAR+ leukemia in all treated mice (figure 6B shows bioluminescence overtime for five mice/cohort, whereas bioluminescence averages were reported in figure 6C). The mouse cohort treated with CAR.CD19 T-cells reached 100% disease-free survival (DFS) at the end of the experimental procedure (day+21) vs 0% DFS for the control cohort of mice receiving NT T-cells. Moreover, we also corroborate the in vitro data relative to NALM-6 CAR.CD19_LL/SH. Also in the in vivo setting, CAR T-cells were able to exert antileukemia control against NALM-6 CAR.CD19_LL/SH (figure 5G-I), although to a lower extent of those observed in CAR.CD19_SL/LH model (figure 5D-F and online supplemental figure 10).