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

Abstract

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.

Introduction

Chimeric antigen receptor T cell (CAR-T) immunotherapy has been a great success in the treatment of liquid cancers, providing rapid and durable responses^1,2,3,4,5. However, disease relapse often occurs in these patients. The main reasons for relapse are either CAR-specific antigen loss on cancerous cells or poor performance of CAR-T cells due to exhaustion or decreased persistence^{6,7,8,9,10,11,12}. Antigen escape has been reported not only for CD19-directed CARs, but also for BCMA¹³, EGFRvIII¹⁴, and IL13Rα2¹⁵, highlighting the drawback of CARs targeting a single tumor-associated antigen. Thus, there is a great need to identify alternative targeting strategies and CAR designs¹⁶. Targeting multiple markers on malignant cells is a promising method to combat antigen escape⁶. Due to the inherent binding nature of naturally occurring ligands, CARs composed of a ligand or receptor ectodomain may have the ability to bind multiple proteins. A small number of ligand- or receptor-based CARs have been tested clinically and have shown encouraging results^17,18, presenting an intriguing design approach to combat antigen escape.

Most CAR-T therapies are focused on singular targeting of CD19 or other limited antigens¹⁹. Such concentrated effort on a few targets may distract future innovation by diluting resources and participation of patients in clinical trials, thus urgently warranting identification and development of new CAR targets. One attractive target is the set of B-cell activating factor (BAFF) receptors. BAFF ligand is a critical B cell survival factor that binds three receptors: BAFF-R, TACI, and BCMA²⁰. These receptors are expressed by mature B cells and in a wide range of B cell neoplasms^{21,22,23,24,25}. The ability of BAFF to bind to multiple receptors may protect against antigen escape due to the decreased likelihood that cancerous B cells can evade cytotoxic CAR-Ts by downregulating BAFF receptors, since they are important for cell survival. Furthermore, in contrast to approved therapies directed against the pan-B cell marker CD19, CAR-Ts designed to target BAFF receptors may be considered a more selective approach to eliminate malignant B cells, due to their more limited expression during B cell development. Currently, the production of CAR-Ts is primarily achieved using viral transduction of transgenes into primary human T cells²⁶.

Despite the relative success of viral transduction, the manufacturing of human CAR-T products is expensive, complex, and associated with notable safety considerations, supporting the need for alternative methods^27,28,29. Since the first human application of transposon-mediated gene therapy almost 10 years ago³⁰, a dozen more clinical trials are underway or have been completed, supporting transposon-based systems as a safe and stable gene transfer alternative with comparable or superior efficiencies to viral transduction³¹.

Here, we report the development and validation of a BAFF ligand-based CAR-T cell product, which can be successfully generated using the non-viral TcBuster (TcB) transposon system. We show that these BAFF CAR-T cells are both functional and specific in targeting the three BAFF receptors (BAFF-R, BCMA, and TACI) expressed by multiple B cell cancers. We demonstrate robust in vitro and in vivo cytotoxicity exerted by BAFF CAR-T cells against mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL) xenograft mouse models.

Results

A BAFF ligand-based CAR-T developed using non-viral gene delivery

We designed the BAFF ligand-based CAR construct using intracellular CD28, OX40/CD134 costimulatory domains, and the CD3ζ signaling domain, as well as the CD28 transmembrane domain (Fig. 1a). To effectively bind the BAFF receptors, we used a truncated BAFF sequence that encompasses the majority of the extracellular domain of the natural human BAFF ligand. We also created a construct that lacks BAFF but is identical to the rest of the BAFF-CAR construct to serve as a negative control (no-BAFF control) (Fig. 1a). Due to the homology between soluble endogenous human BAFF and the extracellular domain of our BAFF-CAR, it is reasonable to predict that BAFF CAR-Ts will bind to the classical BAFF receptors BAFF-R, BCMA, and TACI, thus conferring multi-antigen specificity. Importantly, we also utilize a non-viral genetic engineering approach to stably integrate the BAFF-CAR into T cells using the TcB transposon system. Following co-electroporation of TcB transposase-encoding mRNA and transposon plasmid into T cells, the transposase enzyme excises the BAFF-CAR “cargo” from the transposon plasmid before integrating it into genomic DNA, from which BAFF-CAR protein is stably expressed (Fig. 1b). For traditional lentiviral transduction, a modified pLVX expression vector was used (Supplementary Fig. 1a). Both lentiviral and TcB-based delivery resulted in stable BAFF-CAR expression, as evidenced by the detection of BAFF ligand on the T cell surface via flow cytometry (Fig. 1c). Surface BAFF expression directly correlates with GFP expression in the lentiviral transduced T cells, as the pLVX vector encodes GFP downstream of the same promoter as the BAFF-CAR construct. The CD4/CD8 ratios of donor T cells were also characterized (Fig. 1c). T cells transduced with the no-BAFF control construct express GFP, but not surface BAFF (Supplementary Fig. 1b). BAFF CAR-T cells produced using the TcB transposon method were also assessed for stable expression of BAFF-CAR. After 7 days of expansion, BAFF CAR-T cells produced from different donors were frozen, then thawed and cultured for 21 days. CAR-T cells at 21 days post-thaw expressed a similar amount of cell surface BAFF as those 5 days post-thaw (Supplementary Fig. 1c)….