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
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.
Objective:
Integrins are heterodimeric receptors that convey cell-to-cell and cell-to-matrix interactions. Integrin
αvβ3 is expressed in several tumour entities including melanoma, glioblastoma, breast,
pancreatic and prostate cancer, where it promotes tumour cell survival and metastasis. Here, we generated
αvβ3-specific chimeric antigen receptor (CAR) T-cells and analysed their antitumour
function in pre-clinical models in vitro and in vivo.
Methods:
αvβ3-CARs comprising a super-humanised hLM609 targeting domain with either high or
low affinity (hLM609v7, Kd = 3 nM vs. hLM609v11, Kd = 160 nM) and
equipped with either a long or a short IgG4-Fc extracellular spacer (229 vs. 12 amino acids) were expressed in CD8+
and CD4+ T-cells through lentiviral transduction.
Results:
αvβ3-CAR T-cells eliminated αvβ3-positive
tumour cells rapidly and specifically, produced IFN-γ and IL-2 (CD4+ > CD8+) and exhibited
productive proliferation. In vitro, we observed the strongest reactivity with the higher-affinity hLM609v7
αvβ3-CAR in the short spacer configuration, consistent with the tumour membrane-distal
localization of the hLM609 epitope. In a murine xenograft model of metastatic A-375 melanoma, the strongest antitumour effect
was mediated by the lower-affinity hLM609v11 αvβ3-CAR. Notably, a single administration of
hLM609v11 αvβ3-CAR T-cells was able to induce complete elimination of melanoma lesions,
leading to long-term tumour-free survival.
Conclusions:
These data establish αvβ3 integrin as a novel target for CAR T-cell immunotherapy,
and affirm our previous notion that binding domain affinity and spacer length can be calibrated to augment CAR reactivity.
Clinical implications:
αvβ3-CAR T-cells have therapeutic potential in several prevalent solid tumours,
including melanoma and triple-negative breast cancer.
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