An Innovative PTD-IVT-mRNA Delivery Platform for CAR Immunotherapy of ErbB(+) Solid Tumor Neoplastic Cells

Abstract

Chimeric antigen receptor (CAR) immunotherapy includes the genetic modification of immune cells to carry such a receptor and, thus, recognize cancer cell surface antigens. Viral transfection is currently the preferred method, but it carries the risk of off-target mutagenicity. Other transfection platforms have thus been proposed, such the in vitro transcribed (IVT)-mRNAs. In this study, we exploited our innovative, patented delivery platform to produce protein transduction domain (PTD)-IVT-mRNAs for the expression of CAR on NK-92 cells. CAR T1E-engineered NK-92 cells, harboring the sequence of T1E single-chain fragment variant (scFv) to recognize the ErbB receptor, bearing either CD28 or 4-1BB as co-stimulatory signaling domains, were prepared and assessed for their effectiveness in two different ErbB(+) cancer cell lines. Our results showed that the PTD-IVT-mRNA of CAR was safely transduced and expressed into NK-92 cells. CAR T1E-engineered NK-92 cells provoked high levels of cell death (25–33%) as effector cells against both HSC-3 (oral squamous carcinoma) and MCF-7 (breast metastatic adenocarcinoma) human cells in the co-incubation assays. In conclusion, the application of our novel PTD-IVT-mRNA delivery platform to NK-92 cells gave promising results towards future CAR immunotherapy approaches.

mRNA‐based chimeric antigen receptor T cell therapy: Basic principles, recent advances and future directions

“…81 The novel PTD-IVT-mRNA platform shows potential for mitochondrial disorders and β-thalassemia. 72 In addition, we have also concluded novel therapeutic approaches involving the utilization of mRNA encoding CAR-associated proteins. Famous transposon systems like SB transposon system and PB transposon system promise stable CAR sequence integration, making transitory mRNA-based techniques more permanent.…”

“…PTDs enhance internalization of various cargoes within cells 81 . The novel PTD‐IVT‐mRNA platform shows potential for mitochondrial disorders and β‐thalassemia 72 …”

“…engineered PTD‐mRNA targeting the CAR sequence and delivered it to CAR‐T1E‐NK‐92 cells, enabling them to recognize ErbB receptors. Finally, the results demonstrated that PTD‐mRNA successfully expressed CAR in NK‐92 cells 72 . CAR‐T1E‐engineered NK‐92 cells have shown a high level of target cell death efficiency of 25%–33% in co‐culture trials and have great potential as a treatment against human cells of HSC‐3 (oral squamous cell carcinoma) and MCF‐7 (breast metastatic adenocarcinoma).…”

Recruiting In Vitro Transcribed mRNA against Cancer Immunotherapy: A Contemporary Appraisal of the Current Landscape

“…In our recent work published in 2022, a novel technology has been presented based on PTDs for the transduction of the IVT-mRNAs for anti-ErbB CAR in NK-92 cells [50]. PTDs or CPPs are small-length peptides (usually less than 30 amino acids), with the prototype TAT basic domain (as PTD) derived from HIV-1, able to transverse cellular membranes and at the same time transfer many cargos [208].…”

“…This novel strategy combines the adaptability of peptides with the accuracy of IVT-mRNA, opening a viable path for the development of a next-generation non-viral delivery platform in the area of IVT-mRNA therapeutics. PTD-IVT-mRNAs showed encouraging results for two protein-disease-models, including the mitochondrial disorder fatal infantile cardioencephalomyopathy and COX deficiency (attributed to SCO2 gene mutations) and β-thalassemia [49], as well as in a Chimeric Antigen Receptor (CAR) Immunotherapy of ErbB(+) solid tumor neoplastic cells [50], which are explained thoroughly in the following chapters.…”