ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models

Wallstabe, Lars; Göttlich, Claudia; Nelke, Lena C.; Kühnemundt, Johanna; Schwarz, Thomas; Nerreter, Thomas; Einsele, Hermann; Walles, Heike; Dandekar, Gudrun; Nietzer, Sarah; Hudecek, Michael

doi:10.1172/jci.insight.126345

Cited by 156 publications

(119 citation statements)

References 44 publications

(64 reference statements)

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“…The development of microphysiologic 3D tumor models and their use with CAR T-cells has been described. 10 In brief, collagen scaffolds derived from decellularized porcine gut were fixed in cell crowns and placed in 12-well plates, each well with 2.5 mL medium. 1×10 5 MDA-MB-231_ffluc cells were seeded on the luminal side of the scaffold and cultured for 11 days with complete medium exchanges every 2-3 days and immediately prior to T-cell treatment.…”

Section: Microphysiologic 3d Tumor Modelmentioning

confidence: 99%

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Inhibition of TGF-β-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer

Stüber

Monjezi

Wallstabe

et al. 2020

J Immunother Cancer

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BackgroundImmunotherapy with chimeric antigen receptor (CAR)-engineered T-cells is effective in some hematologic tumors. In solid tumors, however, sustained antitumor responses after CAR T-cell therapy remain to be demonstrated both in the pre-clinical and clinical setting. A perceived barrier to the efficacy of CAR T-cell therapy in solid tumors is the hostile tumor microenvironment where immunosuppressive soluble factors like transforming growth factor (TGF)-β are thought to inhibit the cellular immune response. Here, we analyzed whether CAR T-cells specific for the receptor tyrosine kinase-like orphan receptor 1 (ROR1) antigen, that is frequently expressed in triple-negative breast cancer (TNBC), are susceptible to inhibition by TGF-β and evaluated TGF-β-receptor signaling blockade as a way of neutralizing the inhibitory effect of this cytokine.MethodsCD8+and CD4+ROR1-CAR T-cells were prepared from healthy donors and their antitumor function analyzed using the TNBC cell line MDA-MB-231 in vitro and in a microphysiologic 3D tumor model. Analyses were performed in co-culture assays of ROR1-CAR T-cells and MDA-MB-231 cells with addition of exogenous TGF-β.ResultsThe data show that exposure to TGF-β engages TGF-β-receptor signaling in CD8+and CD4+ROR1-CAR T-cells as evidenced by phosphorylation of small mothers against decapentaplegic homolog 2. In the presence of TGF-β, the cytolytic activity, cytokine production and proliferation of ROR1-CAR T-cells in co-culture with MDA-MB-231 TNBC cells were markedly impaired, and the viability of ROR1-CAR T-cells reduced. Blockade of TGF-β-receptor signaling with the specific kinase inhibitor SD-208 was able to protect CD8+and CD4+ROR1-CAR T-cells from the inhibitory effect of TGF-β, and sustained their antitumor function in vitro and in the microphysiologic 3D tumor model. Combination treatment with SD-208 also led to increased viability and lower expression of PD-1 on ROR1-CAR T-cells at the end of the antitumor response.ConclusionWe demonstrate the TGF-β suppresses the antitumor function of ROR1-CAR T-cells against TNBC in preclinical models. Our study supports the continued preclinical development and the clinical evaluation of combination treatments that shield CAR T-cells from TGF-β, as exemplified by the TGF-β-receptor kinase inhibitor SD-208 in this study.

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Section: Microphysiologic 3d Tumor Modelmentioning

confidence: 99%

“…We sought to evaluate the combination treatment of ROR1-CAR T-cells with SD-208 in a microphysiologic 3D TNBC model (10). In this model, T-cells have to combat a large tumor cell mass and tumor regression can be monitored by BLI over a several-day period.…”

Section: Enhanced Potency Of Ror1-car T-cells In Combination With Tgfmentioning

confidence: 99%

Inhibition of TGF-β-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer

Stüber

Monjezi

Wallstabe

et al. 2020

J Immunother Cancer

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“…In recent years, ROR1 has emerged as a molecule of substantial interest for the development of targeted therapy including mAbs [15], chimeric antigen receptor T-cells (CAR-T) [39,40], bi-specific T-cell engager (BiTE) [41] and SMIs targeting the cytoplasmic tyrosine kinase domain (TKI) [29,42] as well as the extracellular part of ROR1 [43,44]. The common characteristics for these compounds are that effects on signaling are similar.…”

Section: Discussionmentioning

confidence: 99%

ROR1 is Expressed in Diffuse Large B-Cell Lymphoma (DLBCL) and a Small Molecule Inhibitor of ROR1 (KAN0441571C) Induced Apoptosis of Lymphoma Cells

et al. 2020

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The receptor tyrosine kinase ROR1 is absent in most normal adult tissues, but overexpressed in several malignancies. In this study, we explored clinical and functional inhibitory aspects of ROR1 in diffuse large B-cell lymphoma (DLBCL). ROR1 expression in tumor cells was more often observed in primary refractory DLBCL, Richter’s syndrome and transformed follicular lymphoma than in relapsed and non-relapsed DLBCL patients (p < 0.001). A survival effect of ROR1 expression was preliminarily observed in relapsed/refractory patients independent of gender and stage but not of age, cell of origin and international prognostic index. A second generation small molecule ROR1 inhibitor (KAN0441571C) induced apoptosis of ROR1+ DLBCL cell lines, similar to venetoclax (BCL-2 inhibitor) but superior to ibrutinib (BTK inhibitor). The combination of KAN0441571C and venetoclax at EC50 concentrations induced almost complete killing of DLBCL cell lines. Apoptosis was accompanied by the downregulation of BCL-2 and MCL-1 and confirmed by the cleavage of PARP and caspases 3, 8, 9. PI3Kδ/AKT/mTOR (non-canonical Wnt pathway) as well as β-catenin and CK1δ (canonical pathway) were inactivated. In zebra fishes transplanted with a ROR1+ DLBCL cell line, KAN0441571C induced a significant tumor reduction. New drugs with mechanisms of action other than those available for DLBCL are warranted. ROR1 inhibitors might represent a novel promising approach.

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“…Recently, the microphysiological 3D lung cancer and breast cancer models have been created to investigate the antitumor activity of receptor tyrosine kinase‐like orphan receptor 1 (ROR1)‐specific CAR‐T cells, in which the decellularized submucosa and mucosa of porcine jejunum was used as the ECM scaffold for culturing A549 nonsmall cell lung cancer cells and MDA‐MB‐231 cells under static or dynamic culture conditions. [ 160 ] It was demonstrated that the ROR1‐CAR T cells rapidly infiltrated and migrated through the 3D tumor tissues and exhibited a potent antitumor effect, as shown by the secretion of caspase‐cleaved keratin 18, a tumor cell apoptotic marker. Similarly, a 3D microfluidic human ovarian cancer model was developed by embedding human epidermal growth factor receptor 2 (HER2) + ovarian cancer cells (SKOV3) in a micropatterned gelatin methacryloyl (GelMA) hydrogel and micromilled hypoxia device to establish an oxygen gradient across micropatterned cancer cells.…”

Section: Other Relevant Immunocompetent Ooc Modelsmentioning

confidence: 99%

3D Immunocompetent Organ‐on‐a‐Chip Models

2020

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In recent years, engineering of various human tissues in microphysiologically relevant platforms, known as organs‐on‐chips (OOCs), are explored to establish in vitro tissue models that recapitulate the microenvironments found in native organs and tissues. However, most of these models have overlooked the important roles of immune cells in maintaining tissue homeostasis under physiological conditions and in modulating the tissue microenvironments during pathophysiology. Significantly, gradual progress is being made in the development of more sophisticated microphysiologically relevant human‐based OOC models that allow the studies of the key biophysiological aspects of specific tissues or organs, interactions between cells (parenchymal, vascular, and immune cells) and their extracellular matrix molecules, effects of native tissue architectures (geometry, dynamic flow, or mechanical forces) on tissue functions, as well as unravelling the mechanism underlying tissue‐specific diseases and drug testing. In this progress report, the different components of the immune system, as well as immune OOC platforms and immunocompetent OOC approaches that have simulated one or more components of the immune system, are discussed. The challenges to recreate a fully functional tissue system in vitro with a focus on the incorporation of the immune system are also outlined.

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ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models

Abstract: Conflict of interest: MH is an inventor on patents related to chimeric antigen receptor technologies that have been filed by the Fred Hutchinson Cancer Research Center

Cited by 156 publications

References 44 publications

Inhibition of TGF-β-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer

Inhibition of TGF-β-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer

ROR1 is Expressed in Diffuse Large B-Cell Lymphoma (DLBCL) and a Small Molecule Inhibitor of ROR1 (KAN0441571C) Induced Apoptosis of Lymphoma Cells

3D Immunocompetent Organ‐on‐a‐Chip Models

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