PD-L1 CAR effector cells induce self-amplifying cytotoxic effects against target cells

Abstract

BackgroundImmune checkpoint inhibitors and chimeric antigen receptor (CAR)-based therapies have transformed cancer treatment. Recently, combining these approaches into a strategy of PD-L1-targeted CAR has been proposed to target PD-L1high tumors. Our study provides new information on the efficacy of such an approach against PD-L1low targets.MethodsNew atezolizumab-based PD-L1-targeted CAR was generated and introduced into T, NK, or NK-92 cells. Breast cancer MDA-MB-231 and MCF-7 cell lines or non-malignant cells (HEK293T, HMEC, MCF-10A, or BM-MSC) were used as targets to assess the reactivity or cytotoxic activity of the PD-L1–CAR-bearing immune effector cells. Stimulation with IFNγ or with supernatants from activated CAR T cells were used to induce upregulation of PD-L1 molecule expression on the target cells. HER2–CAR T cells were used for combination with PD-L1–CAR T cells against MCF-7 cells.ResultsPD-L1–CAR effector cells responded vigorously with degranulation and cytokine production to PD-L1high MDA-MB-231 cells, but not to PD-L1low MCF-7 cells. However, in long-term killing assays, both MDA-MB-231 and MCF-7 cells were eliminated by the PD-L1–CAR cells, although with a delay in the case of PD-L1low MCF-7 cells. Notably, the coculture of MCF-7 cells with activated PD-L1–CAR cells led to bystander induction of PD-L1 expression on MCF-7 cells and to the unique self-amplifying effect of the PD-L1–CAR cells. Accordingly, PD-L1–CAR T cells were active not only against MDA-MD-231 and MCF-7-PD-L1 but also against MCF-7-pLVX cells in tumor xenograft models. Importantly, we have also observed potent cytotoxic effects of PD-L1–CAR cells against non-malignant MCF-10A, HMEC, and BM-MSC cells, but not against HEK293T cells that initially did not express PD-L1 and were unresponsive to the stimulation . Finally, we have observed that HER-2–CAR T cells stimulate PD-L1 expression on MCF-7 cells and therefore accelerate the functionality of PD-L1–CAR T cells when used in combination.ConclusionsIn summary, our studies show that CAR-effector cells trigger the expression of PD-L1 on target cells, which in case of PD-L1–CAR results in the unique self-amplification phenomenon. This self-amplifying effect could be responsible for the enhanced cytotoxicity of PD-L1–CAR T cells against both malignant and non-malignant cells and implies extensive caution in introducing PD-L1–CAR strategy into clinical studies.

Vγ2 x PD-L1, a Bispecific Antibody Targeting Both the Vγ2 TCR and PD-L1, Improves the Anti-Tumor Response of Vγ2Vδ2 T Cell

“…In vitro, Vg2 x PD-L1-activated Vg2Vd2 T cells were able to selectively kill tumor cells selectively without killing PD-L1 negative non-malignant cells or normal cells. In fact, the activation, degranulation, and subsequent tumor cell killing mediated by Vg2 x PD-L1 were all dependent on simultaneous binding to Vg2Vd2 T cell and PD-L1 expressing tumor cells, demonstrating the safety of our strategy in comparison to PD-L1 chimeric antigen receptor NK cells (36). In line with these in vitro observations, Vg2 x PD-L1 was found to improve Vg2Vd2 T cell mediated tumor growth inhibition in vivo.…”

The FLRT3-UNC5B checkpoint pathway inhibits T cell–based cancer immunotherapies

“…To test FLRT3 blockade in vivo, the 293T-FLRT3 CDX tumor model [PD-L1 negative ( 36 )] with adoptively transferred PBMCs was tested with FLRT3 mAb NP591, PD-1 mAb, or both. FLRT3 blockade with NP591 significantly reduced tumor growth (Fig.…”

Novel Nanotherapeutics for Cancer Immunotherapy by PD-L1-Aptamer-Functionalized and Fexofenadine-Loaded Albumin Nanoparticles

“…PolyA was used here as a control for PD-L1 aptamer, since it could not bind to a target protein specifically due to a lack of complex 3D structures. PD-L1-positive MDA-MB-231 cells and PD-L1-negative A549 cells were incubated with PDL1-NP-cou6 or PolyA-NP-cou6, and evaluated by fluorescence microscopy [ 42 , 43 , 44 ]. As shown in Figure 5 , PD-L1-positive MDA-MB-231 cells treated with PDL1-NP-cou6 generated stronger fluorescent signals compared with those treated with PolyA-NP-cou6, whereas PD-L1-negative A549 cells treated with both nanoparticles generated weak fluorescence ( Figure 5 ).…”