Toll-like receptor 4 signaling activation domains promote CAR T cell function against solid tumors.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses, there is an exciting prospect of further enhancing CAR functionality. Here, we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-ß (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human T cells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and T cell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model, MyD88 and TLR4 CAR T cells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors.

Mucoadhesive film for oral delivery of vaccines for protection of the respiratory tract.

The delivery of vaccines plays a pivotal role in influencing the strength and longevity of the immune response and controlling reactogenicity. Mucosal immunization, as compared to parenteral vaccination, could offer greater protection against respiratory infections while being less invasive. While oral vaccination has been presumed less effective and believed to target mainly the gastrointestinal tract, trans-buccal delivery using mucoadhesive films (MAF) may allow targeted delivery to the mucosa. Here we present an effective strategy for mucosal delivery of several vaccine platforms incorporated in MAF, including DNA plasmids, viral vectors, and lipid nanoparticles incorporating mRNA (mRNA/LNP). The mRNA/LNP vaccine formulation targeting SARS-CoV-2 as a proof of concept remained stable within MAF consisting of slowly releasing water-soluble polymers and an impermeable backing layer, facilitating enhanced penetration into the oral mucosa. This formulation elicited antibody and cellular responses comparable to the intramuscular injection, but also induced the production of mucosal IgAs, highlighting its efficacy, particularly for use as a booster vaccine and the potential advantage for protection against respiratory infections. The MAF vaccine preparation demonstrates significant advantages, such as efficient delivery, stability, and simple noninvasive administration with the potential to alleviate vaccine hesitancy.

Regulation of CD19 CAR-T cell activation based on an engineered downstream transcription factor.

CAR-T cells present a highly effective therapeutic option for several malignant diseases, based on their ability to recognize the selected tumor surface marker in an MHC-independent manner. This triggers cell activation and cytokine production, resulting in the killing of the cancerous cell presenting markers recognized by the chimeric antigen receptor. CAR-T cells are highly potent serial killers that may cause serious side effects, so their activity needs to be carefully controlled. Here we designed a system to control the proliferation and activation state of CARs based on downstream NFAT transcription factors, whose activity can be regulated via chemically induced heterodimerization systems. Chemical regulators were used to either transiently trigger engineered T cell proliferation or suppress CAR-mediated activation when desired or to enhance activation of CAR-T cells upon engagement of cancer cells, shown also in vivo. Additionally, an efficient sensor to monitor activated CD19 CAR-T cells in vivo was introduced. This implementation in CAR-T cell regulation offers an efficient way for on-demand external control of CAR-T cell activity to improve their safety.

Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing.

The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.