Non-viral expression of chimeric antigen receptors with multiplex gene editing in primary T cells.

Efficient engineering of T cells to express exogenous tumor-targeting receptors such as chimeric antigen receptors (CARs) or T-cell receptors (TCRs) is a key requirement of effective adoptive cell therapy for cancer. Genome editing technologies, such as CRISPR/Cas9, can further alter the functional characteristics of therapeutic T cells through the knockout of genes of interest while knocking in synthetic receptors that can recognize cancer cells. Performing multiple rounds of gene transfer with precise genome editing, termed multiplexing, remains a key challenge, especially for non-viral delivery platforms. Here, we demonstrate the efficient production of primary human T cells incorporating the knockout of three clinically relevant genes (B2M, TRAC, and PD1) along with the non-viral transfection of a CAR targeting disialoganglioside GD2. Multiplexed knockout results in high on-target deletion for all three genes, with low off-target editing and chromosome alterations. Incorporating non-viral delivery to knock in a GD2-CAR resulted in a TRAC-B2M-PD1-deficient GD2 CAR T-cell product with a central memory cell phenotype and high cytotoxicity against GD2-expressing neuroblastoma target cells. Multiplexed gene-editing with non-viral delivery by CRISPR/Cas9 is feasible and safe, with a high potential for rapid and efficient manufacturing of highly potent allogeneic CAR T-cell products.

Safety and immunogenicity of an oral tablet norovirus vaccine, a phase I randomized, placebo-controlled trial.

BACKGROUND: Noroviruses are the leading cause of epidemic acute gastroenteritis and foodborne diarrheal disease in humans. However, there are no approved vaccines for noroviruses. Potential correlates of protection identified through human challenge studies include mucosal IgA, memory B cells, and serum-blocking antibody titers (BT50). METHODS: We conducted a single-site, randomized, double-blind, placebo-controlled clinical trial of an oral norovirus vaccine to determine safety and immunogenicity. This tablet vaccine is comprised of a nonreplicating adenovirus-based vector expressing the VP1 gene from the GI.1 norovirus strain and a double-stranded RNA adjuvant. Sixty-six adult subjects meeting inclusion/exclusion criteria were randomized 2:1 to receive a single vaccine dose or placebo, respectively. Immunogenicity was primarily assessed by serum BT50. Additional outcomes included serum ELISA titers, fecal and saliva antibody titers, memory and antibody-secreting cell (ASC) frequency, and B cell phenotyping. RESULTS: The vaccine was well-tolerated, with no dose-limiting toxicities. Adverse events were mild or moderate. The primary immunological endpoint (increase in BT50 titers) was met in the high-dose group (P = 0.0003), with 78% showing a ≥2-fold rise in titers after a single immunization. Vaccine recipients also developed mucosally primed VP1-specific circulating ASCs, IgA+ memory B cells expressing gut-homing receptor (α4ß7), and fecal IgA, indicating substantial and local responses potentially relevant to prevent norovirus infection. CONCLUSION: This oral norovirus vaccine was well-tolerated and generated substantial immune responses, including systemic and mucosal antibodies as well as memory IgA/IgG. These results are a major step forward for the development of a safe and immunogenic oral norovirus vaccine. TRIAL REGISTRATION: ClinicalTrials.gov NCT02868073. FUNDING: Vaxart.