A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers

Derek P Wong , Nand K Roy , Keman Zhang , Anusha Anukanth, Abhishek Asthana , Nicole J Shirkey-Son , Samantha Dunmire , Bryan J Jones , Walker S Lahr , Beau R Webber , Branden S Moriarity , Paolo Caimi , Reshmi Parameswaran 
Source: Nat Commun.
Publication Date: (2022)
Issue: 13: 217
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Dorsal root gang. (DRG), rat
Species: rat
Tissue Origin: brain
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Species: human
Tissue Origin: kidney
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
4D-Nucleofector® X-Unit

Transfection of TcB transposase mRNA and BAFF-CAR transposon plasmid were achieved using electroporation performed with the 4D-nucleofector (Lonza). Electroporation was performed with the 4D-Nucleofector (Lonza, Basel, Switzerland). T cells were centrifuged to remove the culture medium and resuspended in P3 buffer. For stable BAFF-CAR integration, 2.5 µg of BAFF-CAR transposon backbone. Reactions were set up using the ddPCR Supermix for Probes (no dUTP) (Bio-Rad, Hercules, CA) with 50 ng of genomic DNA per assay and droplets were generated and analyzed with the QX200 Droplet-digital PCR system (Bio-Rad, Hercules, CA).

Pooled CRISPR sgRNA (Synthego) targeting the BAFF receptors (BAFF-R, TACI, BCMA) were introduced into luciferase-expressing Jeko-1 cells or RPMI-8226 cells via nucleofection (Lonza Nucleofector 2b). 1ug of each sgRNA was used in tandem with 1.5 µg of Cas9 mRNA (Sigma-Millipore) into 10e6 cells. Percentage knockout efficiency was measured using flow cytometry 5 days following nucleofection

Primary human cells were sourced from Lonza (Catalog # R-DRG-505),


B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.