CD19/CD20 CAR-T (Gracell Biotechnologies (Shanghai) )

A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy.

We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice.

Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells.

The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

Recent advances in gene therapy have brought novel treatment options for cancer. However, the full potential of this approach has yet to be unlocked due to the limited payload capacity of commonly utilized viral vectors. Virus‐free DNA transposons, including piggyBac , have the potential to obviate these shortcomings. In this study, we improved a previously modified piggyBac system with superior transposition efficiency. We demonstrated that the internal domain sequences (IDS) within the 3′ terminal repeat domain of hyperactive piggyBac ( hyPB ) donor vector contain dominant enhancer elements. Plasmid‐free donor vector devoid of IDS was used in conjunction with a helper plasmid expressing Quantum PBase ™ v2 to generate an optimal piggyBac system, Quantum pBac ™ ( qPB ), for use in T cells. qPB outperformed hyPB in CD20/CD19 CAR‐T production in terms of performance as well as yield of the CAR‐T cells produced. Furthermore, qPB also produced CAR‐T cells with lower donor‐associated variabilities compared to lentiviral vector. Importantly, qPB yielded mainly CD8 + CAR‐T SCM cells, and the qPB‐ produced CAR‐T cells effectively eliminated CD20/CD19‐expressing tumor cells both in vitro and in vivo. Our findings confirm qPB as a promising virus‐free vector system with an enhanced payload capacity to incorporate multiple genes. This highly efficient and potentially safe system will be expected to further advance gene therapy applications.