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Background Wide-spread application of chimeric antigen receptor (CAR) T cell therapy for cancer is limited by the current use of autologous CAR T cells necessitating the manufacture of individualized therapeutic products for each patient. To address this challenge, we have generated an off-the-shelf, allogeneic CAR T cell product for the treatment of glioblastoma (GBM), and present here the feasibility, safety, and therapeutic potential of this approach. Methods We generated for clinical use a healthy-donor derived IL13Rα2-targeted CAR+ (IL13-zetakine+) cytolytic T-lymphocyte (CTL) product genetically engineered using zinc finger nucleases (ZFNs) to permanently disrupt the glucocorticoid receptor (GR) (GRm13Z40-2) and endow resistance to glucocorticoid treatment. In a phase I safety and feasibility trial we evaluated these allogeneic GRm13Z40-2 T cells in combination with intracranial administration of recombinant human IL-2 (rhIL-2; aldesleukin) in six patients with unresectable recurrent GBM that were maintained on systemic dexamethasone (4-12 mg/day). Results The GRm13Z40-2 product displayed dexamethasone-resistant effector activity without evidence for in vitro alloreactivity. Intracranial administration of GRm13Z40-2 in four doses of 108 cells over a two-week period with aldesleukin (9 infusions ranging from 2500–5000 IU) was well tolerated, with indications of transient tumor reduction and/or tumor necrosis at the site of T cell infusion in four of the six treated research subjects. Antibody reactivity against GRm13Z40-2 cells was detected in the serum of only one of the four tested subjects. Conclusions This first-in-human experience establishes a foundation for future adoptive therapy studies using off-the-shelf, zinc-finger modified, and/or glucocorticoid resistant CAR T cells.

Unlimited generation of chimeric antigen receptor (CAR) Tcells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR Tcell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ Tcell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory Tcells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR Tcell characteristics. Expanded iPSC CD19-CAR Tcells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR Tcells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR Tcells also mediated potent antitumor activity invivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR Tcells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

Donor-specific anti-HLA antibodies (DSAs) are a major cause of engraftment failure in patients receiving haploidentical stem cell transplantation (HaploSCT). Effective treatments are needed for these patients, who often have no other donor options and/or are in need to proceed urgently to transplantation. We studied a multimodality treatment with alternate-day plasma exchange (PE), rituximab, intravenous γ globulin (IVIg) and an irradiated donor buffy coat for patients with DSAs at 2 institutions. Thirty-seven patients with a median age of 51 years were treated with this desensitization protocol. Treatment outcomes were compared with a control group of HaploSCT patients without DSAs (n = 345). The majority of patients in the DSA group were female (83.8% vs 37.1% in controls, P < .001) and received stem cells from a child as the donor (67.6% vs 44.1%, P = .002). Mean DSA level before and after desensitization was 10 198 and 5937 mean fluorescence intensity (MFI), respectively, with mean differences of 4030 MFI. Fourteen of 30 tested patients (46.7%) had C1q positivity, while 8 of 29 tested patients (27.6%) remained positive after desensitization. In multivariable analysis, patients with initial DSA > 20 000 MFI and persistent positive C1q after desensitization had a significantly lower engraftment rate, which resulted in significantly higher non-relapse mortality and worse overall survival (OS) than controls, whereas graft outcome and survival of patients with initial DSA < 20 000 MFI and those with negative C1q after treatment were comparable with controls. In conclusion, treatment with PE, rituximab, IVIg, and donor buffy coat is effective in promoting engraftment in patients with DSAs ≤20 000 MFI.

Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity; however, their indirect effects on the endogenous immune system are not well characterized. Remarkably, we demonstrate that CAR T-cell treatment of mouse syngeneic glioblastoma (GBM) activates intratumoral myeloid cells and induces endogenous T-cell memory responses coupled with feed-forward propagation of CAR T-cell responses. IFNγ production by CAR T cells and IFNγ responsiveness of host immune cells are critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these observations is supported by studies showing that human IL13Rα2-CAR T cells activate patient-derived endogenous T cells and monocytes/macrophages through IFNγ signaling and induce the generation of tumor-specific T-cell responses in a responding patient with GBM. These studies establish that CAR T-cell therapy has the potential to shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity. SIGNIFICANCE: Our findings highlight the critical role of IFNγ signaling for a productive CAR T-cell therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T-cell immunity, emphasizing the importance of host innate and adaptive immunity for CAR T-cell therapy of solid tumors.This article is highlighted in the In This Issue feature, p. 2113.

Prevention of graft-versus-host-disease (GVHD) while preserving graft-versus-leukemia/lymphoma (GVL) effect remains the Holy Grail for allogeneic hematopoietic cell transplantation (HCT). Purified donor CD4+ T cells induce strong but CD8+ T cells induce little acute GVHD (aGVHD); however, the mechanisms remain unknown. Recipient tissue expression of PD-L1 (B7H1) down-regulates aGVHD via simultaneous PD-L1 interactions with CD80 and PD-1 on donor T cells, but whether this mechanism impacts on GVL effect remains unclear. In the current studies, with MHC-mismatched HCT model of C57BL/6 donor to BALB/c recipient, we observed that 1) although sorted wild-type (WT) CD8+T cells induced no signs of aGVHD, purified PD-1−/− CD8+ T cells induced lethal aGVHD; 2) while WTCD8+ T or very low-dose (0.1×106) CD4+ T cells alone induced little signs of aGVHD, co-transplantation of them induced lethal aGVHD; 3) depletion of CD4+ T cells by anti-CD4 mAb early after HCT prevented aGVHD in wild-type but not in PD-L1−/− recipients, and the GVHD prevention is associated with increase of serum IFN-γ, upregulation of host tissue PD-L1, upregulation of CD80 and PD-1 by CD8+ T cells, as well as increase of apoptosis and anergy of tissue infiltrating CD8+ T cells; 4) interestingly, the depletion of donor CD4+ T cells augmented donor CD8+T cell proliferation without increasing anergy or apoptosis in the lymphoid tissue and preserved strong GVL effect. These results indicate that depletion of donor CD4+ T cells early after HCT leads to increasing susceptibility of donor CD8+ T cells to host tissue PD-L1-mediated apoptosis and anergy in GVHD target tissues but not in the lymphoid tissues, such that GVHD is prevented and GVL effect is preserved.

In acute graft versus host disease (GVHD), donor CD4+ T cells recognize alloantigens on host antigen-presenting cells and differentiate into T helper (Th) subsets (i.e. Th1, Th2, and Th17), but the role of Th subsets in GVHD pathogenesis is still elusive. We found in an MHC-mismatched model of C57BL/6 donor to BALB/c recipient, wild-type donor CD4+ T cells predominantly differentiate into Th1 and preferentially mediate GVHD tissue damage in gut and liver. However, lack of IFN-γ in donor CD4+ T cells results in augmented Th2 and Th17 differentiation and exacerbated tissue damage in lung and skin; lack of both IL-4 and IFN-γ results in augmented Th17 differentiation and exacerbated tissue damage in skin; and lack of both IFN-γ and IL-17 leads to further augmentation of Th2 differentiation, expansion of TNF-α -producing proinflammatory Th2, and idiopathic pneumonia. The organ-specific GVHD mediated by differential Th subsets is associated with differential expression of chemokine receptors by different Th subsets and different chemokines released by various organ tissues. Furthermore, lack of tissue expression of IFN-γ -inducible co-inhibitory molecule B7-H1 play a critical role in augmenting the Th2-mediated idiopathic pneumonia. These results indicate that donor CD4+ T cells reciprocally differentiate into Th1, Th2, and Th17 in acute GVHD recipients; different Th subsets mediate organ-specific tissue damage.