CD28 Costimulatory Domain-Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function.

An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1 Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.

Generation of Antitumor T Cells For Adoptive Cell Therapy With Artificial Antigen Presenting Cells.

Adoptive cell therapy with ex vivo expanded tumor infiltrating lymphocytes or gene engineering T cells expressing chimeric antigen receptors (CAR) is a promising treatment for cancer patients. This production utilizes T-cell activation and transduction with activation beads and RetroNectin, respectively. However, the high cost of production is an obstacle for the broad clinical application of novel immunotherapeutic cell products. To facilitate production we refined our approach by using artificial antigen presenting cells (aAPCs) with receptors that ligate CD3, CD28, and the CD137 ligand (CD137L or 41BBL), as well as express the heparin binding domain (HBD), which binds virus for gene-transfer. We have used these aAPC for ex vivo gene engineering and expansion of tumor infiltrating lymphocytes and CAR T cells. We found that aAPCs can support efficacious T-cell expansion and transduction. Moreover, aAPCs expanded T cells exhibit higher production of IFN-γ and lower traits of T-cell exhaustion compared with bead expanded T cells. Our results suggest that aAPC provide a more physiological stimulus for T-cell activation than beads that persistently ligate T cells. The use of a renewable cell line to replace 2 critical reagents (beads and retronectin) for CAR T-cell production can significantly reduce the cost of production and make these therapies more accessible to patients.

Genome Sequences of Nine Erwinia amylovora Bacteriophages.

Erwinia amylovora is a plant pathogen belonging to the Enterobacteriaceae family, a family containing many plant and animal pathogens. Herein, we announce nine genome sequences of E. amylovora bacteriophages isolated from infected apple trees along the Wasatch Front in Utah.

Genome Sequences of 19 Novel Erwinia amylovora Bacteriophages.

Erwinia amylovora is the causal agent of fire blight, a devastating disease affecting some plants of the Rosaceae family. We isolated bacteriophages from samples collected from infected apple and pear trees along the Wasatch Front in Utah. We announce 19 high-quality complete genome sequences of E. amylovora bacteriophages.