Plataforma de Nanopartículas Lipídicas Ionizáveis Visando Imunoterapia do Câncer

Abstract

Antitumor immunotherapy is a hallmark in the treatment of cancer. In this context, ionizable lipid nanoparticle (LNP) platforms are alternatives for the delivery of nucleic acids. In the production of CAR T cells, ex vivo, LNPs can be used to induce transient expression of the receptor, whether delivering DNA or mRNA. Thus, they offer an alternative to overcome adverse effects observed with the use of viral vectors. LNPs can also be optimized and functionalized to deliver nucleic acids with high specificity to cells of interest. In addition of being an alternative for the production of CAR T cells in vivo, functionalized LNPs encapsulating mRNA can be used as therapeutic vaccine platforms against cancer, inducing an immune response against tumor antigens. In the present work we addressed different applications of an LNP platform, such as optimization for the delivery of DNA and mRNA for the production of CAR T cells in vitro, and also its functionalization for targeted delivery to myeloid cells. In the first part of this work, we demonstrated that increasing the molar ratio of DOPE favors the delivery of DNA to human T lymphocytes. We identified a formulation capable of transfecting Jurkat cells for anti-CD19 CAR expression. Cells transfected with LNP increased the expression of activation markers (CD69 and PD1) when co-cultured with Raji cells (CD19+). Using this LNP, we transfected primary human T lymphocytes and demonstrated the cytotoxic potential of these cells against CD19+ target cells. We demonstrated that transfection with the LNP optimized in this work was greater in Jurkat cells than transfection with the formulation synthesized with ionizable lipids already approved for use in humans. We used this platform to deliver mRNA for CAR expression against solid tumors, using Claudin 6 as a target (CAR-CLDN6). To achieve this, we synthesized mRNA modified with pseudouridine (ψUTP) and delivered this mRNA to T lymphocytes with the optimized LNP. We demonstrated transfection in Jurkat cells, and subsequent activation in co-culture with Colo-205 (CLDN6+) colorectal carcinoma cells. Primary human T lymphocytes transfected with the LNP encapsulating mRNA for CAR-CLDN6 were effective against CLDN6+ cells. This indicates that the LNP platform can also be used to produce CAR T cells with mRNA. Finally, we functionalized the LNP optimized in the in vitro studies with a CD11b antibody for targeted delivery of OVA mRNA to myeloid cells in vivo. To test the functionalized platform, we used a therapeutic vaccine approach, immunizing animals with LNP prior to inoculation of OVA-expressing melanoma cells (B16F10-OVA). Data from the in vivo study demonstrate that the vaccine induced a cellular response, with activation of lymphocytes and production of cytokines, in addition to inducing an increase in the frequency of myeloid cells presenting MCH-II. Despite not having controlled tumor growth, this therapeutic approach opens up new possibilities for the functionalization and use of LNPs delivering mRNA, requiring further studies to adjust doses and use new specific targets for solid tumors.