RESUMEN
Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 ( NCT03500991 ), an ongoing phase 1 clinical trial at Seattle Children's evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients.
Asunto(s)
Glioblastoma/terapia , Inmunoterapia Adoptiva/efectos adversos , Receptor ErbB-2/genética , Receptores Quiméricos de Antígenos/genética , Antígenos CD19/inmunología , Quimiocina CCL2/genética , Quimiocina CXCL10/genética , Femenino , Glioblastoma/líquido cefalorraquídeo , Glioblastoma/genética , Glioblastoma/inmunología , Humanos , Inmunidad/genética , Inmunidad/inmunología , Estimación de Kaplan-Meier , Masculino , Recurrencia Local de Neoplasia/líquido cefalorraquídeo , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/terapia , Receptor ErbB-2/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/uso terapéutico , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Synthetic immunology, as exemplified by chimeric antigen receptor (CAR) T-cell immunotherapy, has transformed the treatment of relapsed/refractory B cell-lineage malignancies. However, there are substantial barriers-including limited tumor homing, lack of retention of function within a suppressive tumor microenvironment, and antigen heterogeneity/escape-to using this technology to effectively treat solid tumors. A multiplexed engineering approach is needed to equip effector T cells with synthetic countermeasures to overcome these barriers. This, in turn, necessitates combinatorial use of lentiviruses because of the limited payload size of current lentiviral vectors. Accordingly, there is a need for cell-surface human molecular constructs that mark multi-vector cotransduced T cells, to enable their purification ex vivo and their tracking in vivo. To this end, we engineered a cell surface-localizing polypeptide tag based on human HER2, designated HER2t, that was truncated in its extracellular and intracellular domains to eliminate ligand binding and signaling, respectively, and retained the membrane-proximal binding epitope of the HER2-specific mAb trastuzumab. We linked HER2t to CAR coexpression in lentivirally transduced T cells and showed that co-transduction with a second lentivirus expressing our previously described EGFRt tag linked to a second CAR efficiently generated bispecific dual-CAR T cells. Using the same approach, we generated T cells expressing a CAR and a second module, a chimeric cytokine receptor. The HER2txEGFRt multiplexing strategy is now being deployed for the manufacture of CD19xCD22 bispecific CAR T-cell products for the treatment of acute lymphoblastic leukemia (NCT03330691).
Asunto(s)
Inmunoterapia Adoptiva/métodos , Lentivirus/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Vectores Genéticos , Humanos , Ratones , Péptidos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Transducción Genética , Trastuzumab/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cytotoxic chemotherapy and radiation can render lymphocyte repertoires qualitatively and quantitatively defective. Thus, heavily treated patients are often poor candidates for the manufacture of autologous chimeric antigen receptor (CAR)-T cell products. In the United States and Europe, children with high-risk neuroblastoma undergo apheresis early in the course of treatment to collect peripheral blood stem cells (PBSCs) for cryopreservation in preparation for high-dose chemotherapy followed by autologous stem cell rescue. Here, we investigate whether these cryopreserved chemotherapy and granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs can serve as starting material for CAR-T cell manufacturing. We evaluated T cell precursor subsets in cryopreserved PBSC units from 8 patients with neuroblastoma using fluorescent activated cell sorting-based analysis. Every cryopreserved unit collected early in treatment contained both CD4 and CD8 precursors with significant numbers of naïve and central memory precursors. Significant numbers of Ki67+/PD1+ T cells were detected, presumably the result of chemotherapy-induced lymphopenia and subsequent homeostatic proliferation. Cryopreserved PBSC units containing 56 to 112â¯×â¯106 T cells were amenable to immunomagnetic selection, CD3â¯×â¯28 bead activation, lentiviral transduction, and cytokine-driven expansion, provided that CD14 monocytes were depleted before the initiation of cultures. Second- and third-generation CD171 CAR+ CD4 and CD8 effector cells derived from cryopreserved units displayed antineuroblastoma lytic potency and cytokine secretion comparable to those derived from a healthy donor and mediated in vivo antitumor regression in NSG mice. We conclude that cryopreserved PBSCs procured via standard methods during early treatment can serve as an alternative starting source for CAR-T cell manufacturing, extending the options for heavily treated patients.
Asunto(s)
Traslado Adoptivo , Criopreservación , Movilización de Célula Madre Hematopoyética , Neuroblastoma , Células Madre de Sangre Periférica , Receptores Quiméricos de Antígenos/inmunología , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neuroblastoma/inmunología , Neuroblastoma/patología , Neuroblastoma/terapia , Células Madre de Sangre Periférica/inmunología , Células Madre de Sangre Periférica/patología , Estudios Retrospectivos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells.
RESUMEN
PURPOSE: The identification and vetting of cell surface tumor-restricted epitopes for chimeric antigen receptor (CAR)-redirected T-cell immunotherapy is the subject of intensive investigation. We have focused on CD171 (L1-CAM), an abundant cell surface molecule on neuroblastomas and, specifically, on the glycosylation-dependent tumor-specific epitope recognized by the CE7 monoclonal antibody. EXPERIMENTAL DESIGN: CD171 expression was assessed by IHC using CE7 mAb in tumor microarrays of primary, metastatic, and recurrent neuroblastoma, as well as human and rhesus macaque tissue arrays. The safety of targeting the CE7 epitope of CD171 with CE7-CAR T cells was evaluated in a preclinical rhesus macaque trial on the basis of CD171 homology and CE7 cross reactivity. The feasibility of generating bioactive CAR T cells from heavily pretreated pediatric patients with recurrent/refractory disease was assessed. RESULTS: CD171 is uniformly and abundantly expressed by neuroblastoma tumor specimens obtained at diagnoses and relapse independent of patient clinical risk group. CD171 expression in normal tissues is similar in humans and rhesus macaques. Infusion of up to 1 × 108/kg CE7-CAR+ CTLs in rhesus macaques revealed no signs of specific on-target off-tumor toxicity. Manufacturing of lentivirally transduced CD4+ and CD8+ CE7-CAR T-cell products under GMP was successful in 4 out of 5 consecutively enrolled neuroblastoma patients in a phase I study. All four CE7-CAR T-cell products demonstrated in vitro and in vivo antitumor activity. CONCLUSIONS: Our preclinical assessment of the CE7 epitope on CD171 supports its utility and safety as a CAR T-cell target for neuroblastoma immunotherapy. Clin Cancer Res; 23(2); 466-77. ©2016 AACR.
Asunto(s)
Inmunoterapia Adoptiva , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Neuroblastoma/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/inmunología , Línea Celular Tumoral , Epítopos/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Lentivirus/genética , Macaca mulatta , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/terapia , Molécula L1 de Adhesión de Célula Nerviosa/genética , Neuroblastoma/inmunología , Neuroblastoma/patología , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Chimeric antigen receptor (CAR) development is biased toward selecting constructs that elicit the highest magnitude of T-cell functional outputs. Here, we show that components of CAR extracellular spacer and cytoplasmic signaling domain modulate, in a cooperative manner, the magnitude of CD8(+)CTL activation for tumor-cell cytolysis and cytokine secretion. Unexpectedly, CAR constructs that generate the highest in vitro activity, either by extracellular spacer length tuning or by the addition of cytoplasmic signaling modules, exhibit attenuated antitumor potency in vivo, whereas CARs tuned for moderate signaling outputs mediate tumor eradication. Recursive CAR triggering renders CTLs expressing hyperactive CARs highly susceptible to activation-induced cell death (AICD) as a result of augmented FasL expression. CAR tuning using combinations of extracellular spacers and cytoplasmic signaling modules, which limit AICD of CD8(+)CTLs, may be a critical parameter for achieving clinical activity against solid tumors.
Asunto(s)
Neoplasias Encefálicas/terapia , Neuroblastoma/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Neoplasias Encefálicas/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Proteína Ligando Fas/inmunología , Vectores Genéticos , Humanos , Lentivirus/genética , Activación de Linfocitos/inmunología , Ratones Endogámicos NOD , Ratones SCID , Neuroblastoma/inmunología , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/inmunología , Transducción de Señal/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor fas/inmunologíaRESUMEN
Global inactivation of the metalloproteinase ADAM17 during mouse development results in perinatal lethality and abnormalities of the heart, including late embryonic cardiomegaly and thickened semilunar and atrioventricular valves. These defects have been attributed in part to a lack of ADAM17-mediated processing of HB-EGF, as absence of soluble HB-EGF results in similar phenotypes. Because valvular mesenchymal cells are largely derived from cardiac endothelial cells, we generated mice with a floxed Adam17 allele and crossed these animals with Tie2-Cre transgenics to focus on the role of endothelial ADAM17 in valvulogenesis. We find that although hearts from late-stage embryos with ablation of endothelial ADAM17 appear normal, an increase in valve size and cell number is evident, but only in the semilunar cusps. Unlike Hbegf(-/-) valves, ADAM17-null semilunar valves do not differ from controls in acute cell proliferation at embryonic day 14.5 (E14.5), suggesting compensatory processing of HB-EGF. However, levels of the proteoglycan versican are significantly reduced in mutant hearts early in valve remodeling (E12.5). After birth, aortic valve cusps from mutants are not only hyperplastic but also show expansion of the glycosaminoglycan-rich component, with the majority of adults exhibiting aberrant compartmentalization of versican and increased deposition of collagen. The inability of mutant outflow valve precursors to transition into fully mature cusps is associated with decreased postnatal viability, progressive cardiomegaly, and systolic dysfunction. Together, our data indicate that ADAM17 is required in valvular endothelial cells for regulating cell content as well as extracellular matrix composition and organization in semilunar valve remodeling and homeostasis.