Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.
Chimeric antigen receptor (CAR) T-cell immunotherapy has been regarded as one of the most compelling breakthroughs in cancer treatment in recent years. In CAR therapy, the chimeric pairing of an antigen receptor with an intracellular tyrosine-based activation motif from the T-cell receptor allows CD8+ cytotoxic T cells to target surface cell markers with robust yet major histocompatibility-independent activation.1 Consequently, a prerequisite for effective CAR T-cell therapy is the identification of suitable target antigens that allow for ablation of the cancer cells while minimizing off-target effects.1, 2 As of today, current CAR T-cell therapy clinical trials, as well as the major biotech players in the field, focus primarily on B-cell hematological malignancies by targeting CD19.1, 2 However, peripheral T-cell lymphomas (PTCLs) account for 10–15% of all non-Hodgkin’s lymphomas (NHLs) and are more difficult to treat as compared with B-cell NHLs. Indeed, stage-by-stage, with few exceptions, T-cell NHLs have poorer outcomes, lower response rates, shorter times to progression and shorter median survivals as compared with B-cell NHLs.2, 3 As a result, the standard of care for PTCLs is not well established, with the only potential curative regimen being allogeneic bone marrow transplantation (BMT), which in itself has significant treatment-associated mortality.4 Despite this clinical need, preclinical reports of targeting CAR T cells against T-cell malignancies are almost non-existent.1, 2
CD4 antigen is expressed in the majority of mature T-cell lymphomas as well as in a subset of T-cell acute lymphoblastic leukemias. Notably, in CD4+ mature T-cell lymphomas, CD4 expression is uniform across all cells. Because CD4 expression is restricted to the hematopoietic compartment and not expressed in hematopoietic stem cells, targeting CD4 minimizes the risk of off-target effects in non-hematological tissues. In the past 25 years, anti-CD4 antibodies have been extensively used for CD4+ cell depletion in non-human primate models5 as well as in clinical trials for rheumatoid arthritis,6, 7, 8, 9, 10 multiple sclerosis,11, 12, 13 lupus,14 psoriasis,15, 16, 17 as well as cutaneous18, 19 and PTCL.20, 21 Combined, these studies provide detailed data on clinical response, adverse events and toxicity in transient CD4+ cell depletion. Overall, CD4+ cell depletion is reversible and well tolerated, and in many cases, there is no clinical evidence of immunosuppression.18 However, targeting CD4 with antibodies has met only limited success in T-cell malignancies, suggesting that alternative and more potent therapies targeting CD4 are required.18, 22
The purpose of this preclinical study is to determine the efficacy of a CD4-specific CAR (CD4CAR) and therefore evaluate CD4CAR potential to deplete CD4+ T-cell malignancies in patients with minimal residual disease who cannot qualify for BMT, currently the only approved curative option for patients with PTCL. In other words, transient CAR-mediated CD4+ depletion would be used as a short-term 'bridge to transplant' that would allow patients with CD4+ T-cell malignancies to become eligible for BMT, and BMT itself would be preceded by a myeloablative regimen.
CAR T cells contain CD4+ and CD8+ populations and the relative importance of the CD4/CD8 ratio is unknown. The efficacy of adoptive cell therapy is most often attributed to CD8+ T cells.23 Indeed, fully functional memory CD8+ T cells are observed in the absence of CD4+ cells.24 Furthermore, in mice, infusion of CD19 CAR-targeted CD8+ T cells alone was sufficient for long-term B-cell eradication and CAR T-cell persistence.25, 26 CD8+ memory T cells from CAR-modified T cells are now the subject of an Food and Drug Administration (FDA)-authorized clinical trial for the treatment of CD19+ B-cell malignancies.27
In our study, we modified CD8+ T cells to express a third-generation CD4CAR, which incorporates CD28, 4-1BB and CD4zeta signaling moieties. We found that CD4CAR T cells exhibited profound leukemic cell killing ability for the KARPAS 299 CD4+ lymphoma cell line. In co-culture experiments, CD4CAR T cells also effectively eliminated leukemic cells from patients with PTCLs. Furthermore, we demonstrated that CD4CAR T cells displayed significant in vivo antileukemic effects in xenogeneic mouse models. Our results suggest that CD4CAR T cells present a promising immunotherapy approach for the treatment of aggressive PTCLs and other T-cell malignancies.
Materials and methods
Blood donors, primary tumor cells and cell lines
Human lymphoma cells and peripheral blood mononuclear cells (PBMCs) were obtained from residual samples on a protocol approved by the Institutional Review Board of Stony Brook University. Umbilical cord blood (CB) cells were obtained from donors at the Stony Brook University Hospital. Written, informed consent was obtained from all donors. SP53 and KARPAS 299 lymphoma cell lines were obtained from ATCC (Manassas, VA, USA).
Lentivirus production and transduction of T cells
To produce viral supernatant, 293FT cells were co-transfected with pMD2G and pSPAX viral packaging plasmids, and with either pRSC.CD4.3G or green fluorescent protein (GFP) Lentiviral vector, using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) per the manufacturer’s protocol. Prior to lentiviral transduction, umbilical cord or peripheral blood mononuclear buffy coat cells were activated for 2 days in the presence of 300 IU/ml interleukin (IL)-2 and 1 μg/ml anti-human CD3 (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4CAR transfection, transduction and validation of C4CAR expression in T cells are described in detail in Supplementary Information.
CAR-transduced T cells were expanded for 7 days in T-cell media (50% AIM-V, 40% RPMI 1640, 10% fetal bovine serum and 1 × penicillin/streptomycin; all Gibco, Carlsbad, CA, USA) supplemented with IL-2. Cells were counted every day, and media was added every 2–3 days in order to maintain T-cell counts <2 × 106 cells/ml.
For the analysis of CAR cell immunophenotype, following 7 days of expansion, CD4CAR T cells and GFP control cells were stained with CD45RO, CD45RA, CD62L and CD8 (all from BD Biosciences, San Jose, CA, USA) for flow cytometry analysis.
Co-culture target cell ablation assays
CD4CAR T cells or GFP T cells (control) were incubated with target cells at ratios of 2:1, 5:1 and 10:1 (200 000, 500 000 or 1 million effector cells to 100 000 target cells, respectively) in 1 ml T-cell culture media without IL-2 for 24 h. Target cells were KARPAS 299 cells (anaplastic large T-cell lymphoma expressing CD4), leukemia cells from a patient with CD4+ T-cell leukemia—Sezary syndrome—and from a patient with CD4+ PTCL lymphoma. As a negative control, CD4CAR T cells and GFP T cells were also incubated with SP53 (mantle cell lymphoma) cells, which do not express CD4, in the same ratios in 1 ml separate reactions. After 24 h of co-culture, cells were stained with mouse anti-human CD8 and CD4 antibodies. In the experiments with SP53 cells, SP53 cells were labeled with CMTMR (Life Technologies) prior to co-culture with T cells, and T cells were labeled with mouse anti-human CD3 (PerCp) after co-culture incubation.
In vivo mouse xenogenic model
NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) from the Jackson Laboratory (Bar Harbor, ME, USA) were used under a Stony Brook University IACUC-approved protocol. Mice were all male and aged between 8 and 12 weeks. Three sets of in vivo experiments were performed with no blinding. For each set, 10 mice were irradiated with a sublethal (2.5 Gy) dose of gamma irradiation and assigned randomly to the treatment or control group. Twenty-four hours later, mice were given one intradermal injection of 0.5 × 106 or 1.0 × 106 KARPAS 299 cells in order to form a measurable subcutaneous tumor within 7 days. Tumor size area was measured every other day. In the first set, 3 days after the injection of 1 million KARPAS 299 cells, 2 million CD4CAR T (5 mice) or 2 million GFP T control cells (5 mice) were administered to the mice intravenously (by tail-vein injection). A second dose of 8 million cells was injected intravenously on day 22. In the second set, 10 NSG mice was irradiated and injected with 0.5 × 106 KARPAS 299 cells. On day 2, mice were injected intravenously with one course of 8 million CD4CAR T cells (5 mice) and 8 million GFP T control cells (5 mice). A second dose of 5.5 million cells was injected intravenously on day 10. In the third set, 10 NSG mice were irradiated and injected with 0.5 × 106 KARPAS 299 cells. On day 1, mice were intravenously injected with 2.5 × 106 CD4CAR T cells or with GFP T control cells (5 mice per group). Intravenous injections were repeated every 5 days for a total of four courses.
Generation of the third-generation CD4CAR
The scFv (single-chain variable fragment) nucleotide sequence of the anti-CD4 molecule was derived from humanized monoclonal ibalizumab (also known as Hu5A8 or TNX-355).28, 29, 30 This monoclonal antibody has been used in Phase I and II clinical trials for blocking HIV binding to CD4.28, 29, 30 To improve signal transduction through the CD4CAR, the intracellular domains of CD28 and 4-1BB co-activators were fused to the CD3zeta T-cell activation signaling domain. Additionally, the leader sequence of CD8 was introduced for efficient expression of the CD4CAR molecule on the cell surface. Indeed, the anti-CD4 scFv is linked to the intracellular signaling domains by a CD8-derived hinge (H) and transmembrane (TM) regions (Figure 1a). The CD4CAR DNA molecule was sub-cloned into a lentiviral plasmid. Because of the presence of two co-activation domains (CD28 and 4-1BB), CD4CAR is considered to be a third-generation CAR. CD4CAR expression is controlled under a strong SFFV (spleen focus-forming virus) promoter and is well suited for hematological applications.31
Characterization of CD4CAR
In order to verify the CD4CAR construct, transfected 293-FT cells were subjected to western blotting analysis. Immunoblotting with an anti-CD3zeta monoclonal antibody showed bands of predicted size for the CD4CAR CD3zeta fusion protein (Figure 1b). As expected, no CD3zeta expression was observed for the GFP control vector (Figure 1b). The generated CD4CAR lentiviruses were also tested for transduction efficiency in HEK293 cells via flow cytometry for scFv (Supplementary Figure S1). Therefore, we confirmed that our generated third-generation CD4CAR contained the CD3zeta intracellular domain on the intracellular end and the scFv on the extracellular end, implying that all other elements were present: CD8 hinge and transmembrane domains, and CD28 and 4-1BB co-activation domains (Figure 1c). For preclinical characterization of CD4CAR expression and function in T cells, human T cells were activated with anti-CD3 antibodies and IL-2, and then transduced, respectively, with CD4CAR and GFP control lentiviral supernatants. The T cells were then expanded for 7 days after transduction.
CB-derived CD4CAR T cells are highly enriched for CD8+ T cells and most of them bear a central memory T-cell-like immunophenotype
Human umbilical CB is an alternate source for allogeneic T-cell therapy.32 Human CB buffy coat cells were activated and transduced with either CD4CAR or control (GFP) lentiviruses. After transduction, both CD4CAR and GFP T cells were expanded for 7 days, with a 20-fold increase in cell count observed for both CD4CAR and GFP T cells (Supplementary Figure S2). At day 7, cells were analyzed by flow cytometry for T-cell subsets (Figure 2a). Flow cytometry analysis showed that ~54% of T cells expressed the CD4CAR (Figure 2b). Furthermore, we analyzed the CD4 and CD8 subsets during the course of T expansion following CD4CAR transduction. Consistent with previous findings,33, 34 a small subset of CD8 cells was induced to express CD4 during T-cell activation with anti-CD3 and costimulatory molecules (Figure 2c). As expected, the CD4+ T subset was almost completely depleted within 3 or 4 days following CD4CAR transduction as compared with GFP control, in which ~33% of cells remained CD4+ (Figure 2c). These data indicate that CD4CAR T cells exhibit potent anti-CD4 activity in vitro during T-cell expansion.
We also evaluated the immunophenotype of CD4CAR T cells at the end of each culture. Following stimulation, naive T cells lose CD45RA and gain CD45RO in order to become central memory T cells. Flow cytometry analysis from three representative experiments showed that 96% of the expanded T cells were CD45RO+, ~83% were CD62L+ and ~80% were CD8+CD45RO+CD62L+, whereas <4% were CD45RA+ (Figure 2d). The CD8+CD45RO+CD62L+ immunophenotype is consistent with the acquisition of a central memory-like phenotype, and low CD45RA+ expression confirms loss of naive T-cell status.35
CD4CAR T cells derived from CB specifically kill CD4-expressing tumor cells
CD4CAR T cells highly enriched for CD8+ T cells were generated (Figure 2c). The cells were then tested in vitro for antileukemic functions using the KARPAS 299 cell line. The KARPAS 299 cell line was initially established from the peripheral blood of a patient with anaplastic large T-cell lymphoma expressing CD4. Cytogenetic analysis has previously shown that KARPAS 299 cells have many cytogenetic abnormalities.36 During co-culture experiments, CD4CAR cells exhibited profound leukemic cell-killing abilities (Figure 3a). First, CB-derived CD4CAR T cells were tested for their ability to ablate KARPAS 299 cells. Indeed, at 24 h incubation and at a low E:T (effector: target) ratio of 2:1, CD4CAR cells successfully eliminated KARPAS 299 cells. As a control, CD4CAR T cells were also tested for their ability to ablate CD4-negative lymphoma cells. SP53 mantle cell lymphoma cell line is a human B-cell lymphoma cell line that does not express CD4. Flow cytometry analysis showed that CD4CAR T cells were unable to lyse or eliminate SP53 mantle cell lymphoma (Figure 3d).
Studies were also conducted using patient samples. Patient 1 presented with an aggressive form of CD4+ T-cell leukemia, Sezary syndrome, which did not respond to standard chemotherapy. Patient 2 presented with an unspecified CD4+ PTCL lymphoma. Flow cytometry analysis of both patient samples revealed strong and uniform CD4 expression, with almost all leukemic cells expressing CD4 (Figures 3b and c). As visualized by flow cytometry analysis, co-culture of patient samples with CD4CAR for 24 h resulted in rapid and definitive ablation of CD4+ malignancies, with, once again, approximately 98% ablation observed for both Sezary syndrome and PTCL co-cultures, consistent with the ablation of KARPAS previously shown (Figures 3b and c). Therefore, we show that, in a co-culture assay, CD4CAR T cells efficiently eliminate two different types of aggressive CD4+ lymphoma/leukemia cells directly from patient samples even at the low E:T ratio of 2:1 (Figures 3b and c). These data support that CD4 is a promising therapeutic target for CD4-positive T-cell leukemias and lymphomas, analogous to the role of CD19 in the targeting of B-cell malignancies via anti-CD19 CAR. Therefore, our patient sample and CD4CAR co-culture assay extends the notion of using CAR to target CD4-positive malignancies.
CD4CAR T cells derived from PBMCs specifically kill CD4 expressing the tumor cell line
As autologous adoptive CAR T therapy is commonly used in the clinic, we then tested CD4CAR T cells derived from PBMCs. PBMCs were activated and transduced with CD4CAR lentiviruses. The CD4 and CD8 sets were monitored by flow cytometry during cell expansion and compared with that of cells transduced with control GFP. The PBMC-derived CD4CAR T cells were highly enriched for CD8+ T cells as observed with CD4CAR T cells derived from CB (Figure 4a), indicative of the role of CD4CAR in the depletion of CD4+. PBMC-derived CD4CAR cells were subsequently tested in their ability to ablate CD4-positive leukemia/lymphoma cells, using the KARPAS 299 cell line. The ablation assay involved the co-culture of CD4CAR T cells or GFP T cells, with KARPAS 299 cells, and with the SP53 mantle cell lymphoma cell line negative control. Reactions were stopped after 24 h: dead cells were stained with 7-AAD (7-aminoactinomycin D) and live cells were analyzed by flow cytometry.37 KARPAS 299 cells incubated with CD4CAR T cells overnight were eliminated at a rate of 38, 62 and 85%, at E:T ratios of 2:1, 5:1 and 10:1, respectively (Figure 4b). Combined, these data demonstrate a strong dose–response relationship. When target cells were incubated with GFP control T cells, no killing of KARPAS 299 cells was observed. These results demonstrate that CD4CAR T-cell ablation is specific to CD4+ targeting.
CD4CAR T cells exhibit significant antitumor activity in vivo
In order to evaluate in vivo antitumor activities, we developed a xenogeneic mouse model using the KARPAS 299 cell line. Multiple different settings were used to test CD4CAR T-cell efficacy in vivo. We first tested ability of the CD4CAR T cells to delay the appearance of leukemia in the NSG mice with a single low dose. Prior to the injection, modified T cells displayed ~40–50% of cells expressing CD4CAR as demonstrated by flow cytometry analysis. Mice received intradermal injections of KARPAS 299 cells, and then a low dose (2 million) of single systemic injection (intravenous administration) of CD4CAR T cells was given. A single low dose of systemic CD4CART cells administration to leukemia-bearing mice caused only transient regression or delayed the appearance of leukemic mass (Figure 5a). When leukemia growth started to accelerate, an additional course of administration of 8 × 106 CD4CAR T cells remarkably arrested the leukemic growth (Figure 5a).
To further test the efficacy of CD4CAR antileukemia activity, we administered two courses of relatively large doses of CD4CAR T cells. Similarly, two injections totaling 13.5 × 106 CD4CAR T cells caused more pronounced leukemia growth arrest as compared with a lower CD4CAR dose but eventually the leukemic cell population recovered (Figure 5b). Finally, we investigated the efficacy of multiple course injections of a low dose of CD4CAR T cells (each 2.5 × 106 cells). We treated the mice bearing subcutaneous leukemia with repeat intravenous injections of CD4CAR T cells, once every 4 or 5 days for total of four injections. After four courses of CD4CAR T-cell administration, one of the four treated mice was tumor free and exhibited no toxic appearance. Multiple-dose CD4CAR T-cell-treated mice displayed more significant antileukemic effect compared with single dose (Figures 5a and c). Moreover, treatment with CD4CAR T cells significantly prolonged the survival of mice bearing KARPAS 299 lymphoma as compared with treatment with the GFP-transduced control T cells (Figure 5d).
The remarkable results in B-cell leukemia/lymphoma patients treated with CAR-engineered T-cells have caused excitement to many specialists treating hematological malignancies. Although CAR T cells for B-cell malignancies are the most advanced in terms of clinical testing, CAR T-cell technology is positioned to expand to other hematological and non-hematological malignancies, especially as new targets are discovered.
We chose to investigate CD4 antigen as a possible target for CAR-expressing T cells as most mature T-cell lymphomas are CD4 positive. CD4 is universally expressed on these lymphoma cells and its expression is absent in hematopoietic stem cells and non-hematopoietic cells. Targeting CD4 with monoclonal antibodies has been widely used in clinical trials for a variety of autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, psoriasis and multiple sclerosis.12, 15, 16, 38, 39, 40, 41 These studies provide invaluable information regarding the safety issue of targeting CD4. Transient depletion of CD4 in mice, non-human primates and humans has proven safe but induces susceptibility to viral infections.18 In addition, the scFv nucleotide sequence of the anti-CD4 molecule used for the generation of CD4CAR T cells was derived from monoclonal ibalizumab (also known as Hu5A8 or TNX-355, a potent and broad HIV-1-neutralizing antibody) and has been used in clinical trials28, 29, 30 for blocking HIV binding to CD4 and therefore has clinically validated affinity for the CD4 antigen.
The expression of CD4CAR on CD8-positive cells likely causes 'on-target' toxicities observed for other CARs, such as CD19CAR with B-cell aplasia. The B-cell aplasia is reversible and prolonged B-cell aplasia is not observed in the clinical trials.42 The 'on-target toxicity' can be avoided if CD4CAR is rapidly eliminated by the activation of a suicide gene and/or if the CD4CAR is only transiently expressed by CD8 cells, such as using an mRNA transfection approach. However, even if both approaches increase safety, they abolish the relative long-term immune control of a disease, which is a major advantage of adoptive T-cell therapy. Some degree of persistent CAR engraftment is required for efficacy, and persistence for at least several months will probably be required based on clinical trials.43 A several month persistence window of CD4 lymphopenia is likely to be a transient effect and might be tolerated in patients based on clinical trial studies with anti-CD4 monoclonal antibodies for autoimmune disorders or T-cell lymphoma and patients with HIV associated with depletion of CD4 cells.44,45,46 In non-human primates, CD4+ cell depression or depletion by anti-CD4 antibodies is reversible.5 CD4CAR T cells could be a useful bridge to bone marrow transplant by achieving complete remission for patients who have minimal residual disease and are no longer responding to chemotherapy. Alternatively, administration of CD4CAR T cells to eliminate leukemic cells followed by bone marrow stem cell rescue to support CD4 lymphopenia could be an appropriate approach for the treatment of aggressive T-cell hematological malignancies.
CAR T cells are comprised of CD4+ and CD8+ populations. Recent studies have shown that memory CD8+ cells are the only cell type contributing to engraftment, and these cells have been considered as the 'active ingredient' for CARs.47 CD8+ memory T cells from CAR-modified T cells are now the subject of an FDA-authorized clinical trial for the treatment of CD19+ B-cell malignancies.27 Notably, most of our CD4CAR T cells had a central memory-like phenotype (CD8+CD45RO+CD62L+) at the end of each culture (Figure 2d).
CAR therapy combines the specificity of an antibody with unhindered CD8+ T-cell cytotoxic activity, and our data strongly suggest that CD4CAR would be efficacious in the treatment of aggressive PTCLs. Numerous studies in NSG mice bearing human cancers have demonstrated the efficacy of the CAR T-cell approach. However, most of these studies result in temporary growth arrest of the grafted tumors because the mouse niche may not be optimal for human T-cell expansion48 and the issue of human T-cell homing in a mouse environment must also be considered.49 In general, CAR T cells perform better in humans than they do in mice. In vivo models of mice bearing xenografts of human malignancies do not recapitulate the human microenvironment, which allows for 1000- to 10 000-fold CAR T-cell expansion in the blood of patients during the first month after infusion.50 Nonetheless, studying the antitumor response by CAR T cells in an animal model is still an important step for assessment of CAR constructs, in terms of efficacy and elaborating future cancer treatment protocols. To that end, we have repeated our xenograft in vivo models three times for rigor. Our in vivo data indicate that repeat administrations of CD4CAR T cells might be a realistic option for the treatment of T-cell malignancies in the clinical setting, which is consistent with previous studies in other types of tumors.51
CD4+ T-cell leukemias and lymphomas are highly aggressive cancers that are notoriously refractory to pharmacotherapy, with no established standard of care. CD4 is a promising target for CAR T cells. We describe a lentiviral vector encoding CD4CAR that specifically recognizes cells expressing CD4. The efficacy and safety of adoptive transfer of CD4CAR T cells as an approach for aggressive T-cell hematological malignancies will need to be further evaluated in a clinical trial setting.
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Yupo Ma is a founder and Chairman of iCell Gene Therapeutics, LLC. The other authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Leukemia website
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Pinz, K., Liu, H., Golightly, M. et al. Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells. Leukemia 30, 701–707 (2016). https://doi.org/10.1038/leu.2015.311
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