No abstract

The ability to read, write, and edit genomic information in living organisms can have a profound impact on research, health, economic, and environmental issues. The CRISPR/Cas system, recently discovered as an adaptive immune system in prokaryotes, has revolutionized the ease and throughput of genome editing in mammalian cells and has proved itself indispensable to the engineering of immune cells and identification of novel immune mechanisms. In this review, we summarize the CRISPR/Cas9 system and the history of its discovery and optimization. We then focus on engineering T cells and other types of immune cells, with emphasis on therapeutic applications. Last, we describe the different modifications of Cas9 and their recent applications in the genome-wide screening of immune cells.

Chimeric antigen receptor (CAR) T cells, which express a synthetic receptor engineered to target specific antigens, have demonstrated remarkable potential to treat haematological malignancies. However, their transition beyond haematological malignancy has so far been unsatisfactory. Here, we discuss recent challenges and improvements for CAR T cell therapy against solid tumors: Antigen heterogeneity which provides an effective escape mechanism against conventional mono-antigen-specific CAR T cells; and the immunosuppressive tumor microenvironment which provides physical and molecular barriers that respectively prevent T cell infiltration and drive T cell dysfunction and hypoproliferation. Further, we discuss the application of CAR T cells in infectious disease and autoimmunity.

BackgroundThe incorporation of co-stimulatory signaling domains into second-generation chimeric antigen receptors (CARs) significantly enhances the proliferation and persistence of CAR-T cells in vivo, leading to successful clinical outcomes.MethodsTo achieve such functional enhancement in transgenic T-cell receptor-engineered T-cell (TCR-T) therapy, we designed a second-generation TCR-T cell in which CD3ζ genes modified to contain the intracellular domain (ICD) of the 4-1BB receptor were selectively inserted into theCD247locus.ResultsThis modification enabled the simultaneous recruitment of key adaptor molecules for signals 1 and 2 on TCR engagement. However, the addition of full-length 4-1BB ICD unexpectedly impaired the expression and signaling of TCRs, leading to suboptimal antitumor activity of the resulting TCR-T cells in vivo. We found that the basic-rich motif (BRM) in the 4-1BB ICD was responsible for the undesirable outcomes, and that fusion of minimal tumor necrosis factor receptor-associated factor (TRAF)-binding motifs at the C-terminus of CD3ζ (zBBΔBRM) was sufficient to recruit TRAF2, the key adaptor molecule in 4-1BB signaling, while retaining the expression and proximal signaling of the transgenic TCR. Consequently, TCR-T cells expressing zBBΔBRMexhibited improved persistence and expansion in vitro and in vivo, resulting in superior antitumor activity in a mouse xenograft model.ConclusionsOur findings offer a promising strategy for improving the intracellular signaling of TCR-T cells and their application in treating solid tumors.

CD19 targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, recent clinical data indicate that a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors on activated CAR T cells. Here, we report a lentiviral two in one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19 targeting CAR T cells in the context of four different checkpoint combinations PD1/TIM3, PD1/LAG3, PD1/CTLA4 and PD1/TIGIT and found that CAR T with PD1/TIGIT downregulation uniquely exerted synergistic antitumor effects in mouse xenograft models compared with PD1 downregulation only, and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analyses of CAR T cells as well as analyses of transcriptomic profiles suggested that downregulation of PD1 enhances short term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD1/TIGIT downregulated CAR T cells generated from diffuse large B cell lymphoma patient derived T cells using a clinically applicable manufacturing process also showed robust antitumor activity and significantly improved persistence in vivo compared with conventional CD19 targeting CAR T cells. Overall, our results demonstrate that the cell intrinsic PD1/TIGIT dual downregulation strategy may prove effective in overcoming immune checkpoint mediated resistance in CAR T therapy.