RESUMO
Targeted delivery of nucleic acid therapeutics to the lungs could transform treatment options for pulmonary disease. We have previously developed oligomeric charge-altering releasable transporters (CARTs) for in vivo mRNA transfection and demonstrated their efficacy for use in mRNA-based cancer vaccination and local immunomodulatory therapies against murine tumors. While our previously reported glycine-based CART-mRNA complexes (G-CARTs/mRNA) show selective protein expression in the spleen (mouse, >99%), here, we report a new lysine-derived CART-mRNA complex (K-CART/mRNA) that, without additives or targeting ligands, shows selective protein expression in the lungs (mouse, >90%) following systemic IV administration. We further show that by delivering siRNA using the K-CART, we can significantly decrease expression of a lung-localized reporter protein. Blood chemistry and organ pathology studies demonstrate that K-CARTs are safe and well-tolerated. We report on the new step economical, organocatalytic synthesis (two steps) of functionalized polyesters and oligo-carbonate-co-α-aminoester K-CARTs from simple amino acid and lipid-based monomers. The ability to direct protein expression selectively in the spleen or lungs by simple, modular changes to the CART structure opens fundamentally new opportunities in research and gene therapy.
RESUMO
Chimeric antigen receptor (CAR) natural killer (NK) cells are an emerging cell therapy with promising results in oncology trials. However, primary human NK cells are difficult to transfect, hampering both mechanistic studies and clinical applications of NK cells. Currently, NK cell CAR modification relies on viral vectors or cell activation. The former raises cost and tolerability issues, while the latter alters NK cell biology. Here, we report that readily synthesized and inexpensive nonviral charge-altering releasable transporters (CARTs) efficiently transfect primary human NK cells with messenger RNA without relying on NK cell activation. Compared with electroporation, CARTs transfect NK cells more efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype and function. We use CARTs to generate cytotoxic primary anti-CD19 CAR NK cells, demonstrating this technology can drive clinical applications of NK cells. To our knowledge, CARTs represent the first efficacious transfection technique for resting primary human NK cells that preserves NK cell phenotype and can enable new biological discoveries and therapeutic applications of this understudied lymphocyte subset.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Linhagem Celular Tumoral , Humanos , Imunoterapia , Células Matadoras Naturais , Neoplasias/terapia , Fenótipo , Receptores de Antígenos Quiméricos/genéticaRESUMO
Here, we report on the results of a phase I/II trial (NCT00490529) for patients with mantle cell lymphoma who, having achieved remission after immunochemotherapy, were vaccinated with irradiated, CpG-activated tumor cells. Subsequently, vaccine-primed lymphocytes were collected and reinfused after a standard autologous stem cell transplantation (ASCT). The primary endpoint was detection of minimal residual disease (MRD) within 1 yr after ASCT at the previously validated threshold of ≥1 malignant cell per 10,000 leukocyte equivalents. Of 45 evaluable patients, 40 (89%) were found to be MRD negative, and the MRD-positive patients experienced early subsequent relapse. The vaccination induced antitumor CD8 T cell immune responses in 40% of patients, and these were associated with favorable clinical outcomes. Patients with high tumor PD-L1 expression after in vitro exposure to CpG had inferior outcomes. Vaccination with CpG-stimulated autologous tumor cells followed by the adoptive transfer of vaccine-primed lymphocytes after ASCT is feasible and safe.
Assuntos
Vacinas Anticâncer/imunologia , Imunidade , Linfoma de Célula do Manto/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/efeitos adversos , Linhagem Celular Tumoral , Determinação de Ponto Final , Feminino , Humanos , Memória Imunológica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Neoplasia Residual/imunologia , Oligodesoxirribonucleotídeos , Transplante Autólogo , Resultado do TratamentoRESUMO
In vivo delivery of antigen-encoding mRNA is a promising approach to personalized cancer treatment. The therapeutic efficacy of mRNA vaccines is contingent on safe and efficient gene delivery, biological stability of the mRNA, and the immunological properties of the vaccine. Here we describe the development and evaluation of a versatile and highly efficient mRNA vaccine-delivery system that employs charge-altering releasable transporters (CARTs) to deliver antigen-coding mRNA to antigen-presenting cells (APCs). We demonstrate in human peripheral blood mononuclear cells that CART vaccines can activate a robust antigen-specific immune response against mRNA-encoded viral epitopes. In an established mouse model, we demonstrate that CARTs preferentially target professional APCs in secondary lymphoid organs upon i.v. injections and target local APCs upon s.c. injection. Finally, we show that CARTs coformulated with mRNA and a Toll-like receptor ligand simultaneously transfect and activate target cells to generate an immune response that can treat and cure mice with large, established tumors.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Imunidade Celular , Neoplasias Experimentais/terapia , RNA Mensageiro/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Vacinação , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/patologia , Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/farmacologia , Linhagem Celular Tumoral , Feminino , Células HeLa , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , RNA Mensageiro/genética , RNA Mensageiro/farmacologia , Linfócitos T/patologiaRESUMO
Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.