Targeted multi-epitope switching enables straightforward positive/negative selection of CAR T cells

Abstract

Chimeric antigen receptor (CAR) T cell technology has enabled successfully novel concepts to treat cancer patients, with substantial remission rates in lymphoid malignancies. This cell therapy is based on autologous T lymphocytes that are genetically modified to express a CAR that recognizes tumor-associated antigens and mediates the elimination of the respective tumor cells. Current limitations include laborious manufacturing procedures as well as severe immunological side effects upon administration of CAR T cells. To address these limitations, we integrated RQR8, a multi-epitope molecule harboring a CD34 epitope and two CD20 mimotopes, alongside a CD19-targeting CAR, into the CD52 locus. Using CRISPR-Cas9 and adeno-associated virus-based donor vectors, some 60% of genome-edited T cells were CAR+/CD20+/CD34+/CD52− without further selection. This could be increased to >95% purity after CD34 tag-based positive selection. These epitope-switched CAR T cells retained cell killing competence against CD19+ tumor cells, and were resistant to alemtuzumab (anti-CD52) but sensitive to rituximab (anti-CD20) in complement-dependent cytotoxicity assays. In conclusion, gene editing-based multiple epitope switching represents a promising development with the potential to improve both the manufacturing procedure as well as the clinical safety of CAR T cells.

Cas12a Ultra enables efficient genome editing in human multipotent and pluripotent stem cells

“…HDR-based strategies are generally used to correct disease causing mutations or mediate the targeted integration of transgenes. The HDR machinery relies on the co-delivery of an HDR template that consist of a short single stranded oligonucleotide 23 , a short double stranded DNA 47 , plasmid-based HDR templates 20 , or AAV-based templates 29 . For instance, in T cells AAV6 vectors have been used to target the integration of a chimeric antigen receptor (CAR) into the TRAC locus 48 .…”

“…The CSF2RA HDR template is composed of homology arms of 684 bp and 337 bp, respectively, which are complementary to the region surrounding the target site on exon 3 of the CSF2RA locus, a codon-optimized cDNA encoding exons 3 to 13 followed by a pA signal of bovine growth hormone (BGHpA). Recombinant AAV type 6 vector particles were produced and titers determined by ddPCR as described previously 28,29 .…”

“…Gene editing in T cells and HSPCs was performed as previously described 25,29 with the following modi cations: for T cells, RNPs at a 1:5 ratio (19 pmol of Cas12a and 95 pmol of gRNA) were transferred to 1x10 6 T cells using program EO-115 and Kit P3 (Lonza, Basel, Switzerland, Cat# V4XP-3032) on a 4D-Nucleofector. For HSPCs, 2x10 5 cells were mixed with Cas12a RNPs at a 1:5 ratio (38 pmol of Cas12a and 190 pmol of gRNA), and the RNPs transferred via electroporation (4D-Nucleofector, program CA137, Kit P3).…”

Tuning CARs: recent advances in modulating chimeric antigen receptor (CAR) T cell activity for improved safety, efficacy, and flexibility

“…For example, combining two CD20 mimotopes with a CD34 epitope (called RQR8) and a CD8-derived membrane anchor resulted in a chimeric surface protein that allows for selective cell sorting and on-demand cell depletion with Rituximab [86]. Furthermore, a CD20based safety switch might be combined with a CAR to create an all-in-one CAR (e.g., CubiCAR), which contains a marker for detection and selection, suicide gene, and CAR in a single construct [87,88].…”

Spotlight on gene therapy in Germany

“…In this context, the largescale production of edited T cells has enabled the clinical exploitation of these modified cells to combat HIV infection [6,7] or other T cell-related disorders. Similarly, precise genome editing has been applied to provide additional features to CAR T cells with the goal of improving their safety and manufacturing procedures [8]. Since the inception of gene therapy with integrating viral vectors in 1990, we have witnessed an expanding effort to apply these strategies in HSCs to provide a lifelong solution to diseases of the hematopoietic system.…”