Generation of T-cell-receptor-negative CD8αß-positive CAR T cells from T-cell-derived induced pluripotent stem cells.

The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of 'off-the-shelf' CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR- CD8αß+ CAR T cells that perform similarly to CD8αß+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αß+ T cells for a broad range of immunotherapies.

CAR T-cell immunotherapy of MET-expressing malignant mesothelioma.

Mesothelioma is an incurable cancer for which effective therapies are required. Aberrant MET expression is prevalent in mesothelioma, although targeting using small molecule-based therapeutics has proven disappointing. Chimeric antigen receptors (CARs) couple the HLA-independent binding of a cell surface target to the delivery of a tailored T-cell activating signal. Here, we evaluated the anti-tumor activity of MET re-targeted CAR T-cells against mesothelioma. Using immunohistochemistry, MET was detected in 67% of malignant pleural mesotheliomas, most frequently of epithelioid or biphasic subtype. The presence of MET did not influence patient survival. Candidate MET-specific CARs were engineered in which a CD28+CD3ζ endodomain was fused to one of 3 peptides derived from the N and K1 domains of hepatocyte growth factor (HGF), which represents the minimum MET binding element present in this growth factor. Using an NIH3T3-based artificial antigen-presenting cell system, we found that all 3 candidate CARs demonstrated high specificity for MET. By contrast, these CARs did not mediate T-cell activation upon engagement of other HGF binding partners, namely CD44v6 or heparan sulfate proteoglycans, including Syndecan-1. NK1-targeted CARs demonstrated broadly similar in vitro potency, indicated by destruction of MET-expressing mesothelioma cell lines, accompanied by cytokine release. In vivo anti-tumor activity was demonstrated following intraperitoneal delivery to mice with an established mesothelioma xenograft. Progressive tumor regression occurred without weight loss or other clinical indicators of toxicity. These data confirm the frequent expression of MET in malignant pleural mesothelioma and demonstrate that this can be targeted effectively and safely using a CAR T-cell immunotherapeutic strategy.

Targeting of Aberrant αvß6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells.

Expression of the αvß6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvß6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αß) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvß6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvß6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvß6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin.

Preclinical in vivo modeling of cytokine release syndrome induced by ErbB-retargeted human T cells: identifying a window of therapeutic opportunity?

The ErbB network is dysregulated in many solid tumors. To exploit this, we have developed a chimeric Ag receptor (CAR) named T1E28z that targets several pathogenetically relevant ErbB dimers. T1E28z is coexpressed with a chimeric cytokine receptor named 4αß (combination termed T4), enabling the selective expansion of engineered T cells using IL-4. Human T4(+) T cells exhibit antitumor activity against several ErbB(+) cancer types. However, ErbB receptors are also expressed in several healthy tissues, raising concerns about toxic potential. In this study, we have evaluated safety of T4 immunotherapy in vivo using a SCID beige mouse model. We show that the human T1E28z CAR efficiently recognizes mouse ErbB(+) cells, rendering this species suitable to evaluate preclinical toxicity. Administration of T4(+) T cells using the i.v. or intratumoral routes achieves partial tumor regression without clinical or histopathologic toxicity. In contrast, when delivered i.p., tumor reduction is accompanied by dose-dependent side effects. Toxicity mediated by T4(+) T cells results from target recognition in both tumor and healthy tissues, leading to release of both human (IL-2/IFN-γ) and murine (IL-6) cytokines. In extreme cases, outcome is lethal. Both toxicity and IL-6 release can be ameliorated by prior macrophage depletion, consistent with clinical data that implicate IL-6 in this pathogenic event. These data demonstrate that CAR-induced cytokine release syndrome can be modeled in mice that express target Ag in an appropriate distribution. Furthermore, our findings argue that ErbB-retargeted T cells can achieve therapeutic benefit in the absence of unacceptable toxicity, providing that route of administration and dose are carefully optimized.