Dual targeting of BCMA and SLAMF7 with the CARtein system: chimeric antigen receptors with intein-mediated splicing elicit specific T cell activation against multiple myeloma

Abstract

Introduction: Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy against multiple myeloma (MM). However, several barriers continue to limit the overall effectiveness of this approach, such as high production costs, prolonged manufacturing timelines, safety issues, and the potential for tumor antigen escape due to selective therapeutic pressure. To overcome these challenges, innovative CAR T strategies, such as engineering modular CAR systems, are being explored. These systems utilize adaptor molecules to enable multi-antigen targeting, thereby enhancing specificity, safety, and overall efficiency of CAR T-cell therapy. Notably, CAR T-cells directed against BCMA and SLAMF7 antigens have generated strong and robust antitumor responses in MM therapy.

Methods: To address the limitations of conventional CAR T therapy, we developed a novel modular CAR platform targeted against BCMA and SLAMF7. This was achieved using a split intein-mediated protein splicing mechanism, which allows specific covalent peptide bonds to form between CAR modules. This strategy maintains an almost seamless CAR structure, preserving its overall integrity and functionality. The design of the intein-spliced CAR system (termed "CARtein") was further optimized through advanced protein structure prediction software.

Results: Cells expressing the spliced CARtein constructs, engineered to target BCMA, SLAMF7, or both antigens simultaneously, demonstrated robust and highly specific activation in response to their respective antigens.

Discussion: These results suggest that the CARtein platform is a promising, versatile, and highly specific approach for the modular design and engineering of CARs, enabling multi-antigen targeting while maintaining structural and functional integrity. This modular strategy addresses key limitations of conventional CAR T-cell therapy and may improve both the safety and effectiveness of future MM treatments.

Keywords: B-cell maturation antigen; SLAMF7; chimeric antigen receptor; immunotherapy; inteins; modular CAR; multiple myeloma; protein splicing.

Figure 1

Dual targeting CARtein strategy. (A) Schematic representation of CARtein modules before and after split intein-mediated protein splicing. (B) Representation of Jurkat (JKT) triple parameter reporter (TPR) expressing the final anti-BCMA and anti-SLAMF7 CARtein constructs. JKT TPR, Jurkat-TPR cells.

Figure 2

CARtein modules structure prediction. (A) Folding prediction of anti-BCMA antigen recognition module containing the scFv (Belantamab) fused to the N-terminal part of IMPDH-1 split intein (including the N-intein and N-extein domain) compared to the unmodified anti-BCMA scFv. (B) Structure prediction of anti-SLAMF7 antigen recognition module consisting in the scFv (Elotuzumab) fused to the N-terminal part of IMPDH-1 split intein compared to unmodified anti-SLAMF7 scFv. (C) Structure prediction of the extracellular domain of the Signaling CARtein module (SCM) spaning C-terminal intein IMPDH-1 (including the C-intein and C-extein domain) followed by IgG₁ spacer domains and CD28 TM compared to the SCM without intein. Relevant residues for split intein-mediated protein splicing are shown.

Figure 3

CARtein complexes prediction. (A) Docking of the predicted structures for anti-BCMA antigen recognition and SCM modules, with key residues for intein-mediated protein splicing being shown. (B) Docking of the predicted tridimensional structures of anti-SLAMF7 antigen recognition and SCM modules, with highlighted relevant residues for split intein-mediated protein splicing. (C) Complex prediction of post spliced anti-BCMA CARtein bound to BCMA ECD, compared to a CAR without exteins. scFv residues proximal to the antigen are highlighted. (D) Complex prediction of post-spliced anti-SLAMF7 CARtein bound to SLAMF7 ECD compared to a CAR devoid of residual exteins. Elotuzumab residues proximal to the antigen are shown.

Figure 4

Structural comparison of the CARtein design and other modular CAR architectures in complex with the antigen. (A, B) Predicted structures of unmodified anti-BCMA and anti-SLAMF7 CARs in complex with their respective extracellular domains (ECDs). (C, D) Predicted post-splicing anti-BCMA and SLAMF7 CARtein complexes comprising residual exteins (GGGSIC) fused to the antigen-binding scFvs. (E, F) Predicted SpyTag/SpyCatcher-based modular CAR complexes for anti-BCMA and anti-SLAMF7. (G, H) Predicted anti-BCMA and anti-SLAMF7 SUPRA CAR system-based complexes depending on SYNZIP1-SYNZIP2 coiled-coil dimerization incorporating a 35-aa glycine/serine linker between the scFv and SYNZIP1. All models preserve the same SCM, scFvs, and antigen sequences.

Figure 5

CARtein maturation and expression after split intein-mediated protein splicing. (A) Representative illustration of anti-BCMA and anti-SLAMF7 CARtein modules before and after intein-mediated CARtein splicing in the ER. (B) Illustrative representation of the staining strategy performed for the evaluation of SCM (Streptactin) mature whole CARtein (Protein L) and SLAMF7 CARtein expression. (C) Flow cytometry histogram overlays of SCM and spliced CARtein expressing unpermeabilized Jurkat-TPR cells. (D) Flow cytometry histogram overlay of Elotuzumab staining in SCM and anti-SLAMF7 or Dual CARtein-TPR cells. (E) Histogram overlay of BCMA and SLAMF7 expression in transduced k562 cells with either or both surface antigens. UTD, untransduced cells. JKT TPR, Jurkat-TPR cells.

Figure 6

Activation signaling assay of anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cells co-cultured with k562 cells expressing BCMA, SLAMF7 or both surface proteins. Representative flow cytometry analysis of NFAT (A) and NFκB (B) promoter reporters, or CD69 (C) in CARtein-TPR cells 24h after co-culture with either untransduced (UTD) or CARtein transduced k562 cells in a T:E ratio of 1:1.

Figure 7

Kinetics of anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cell activation upon co-culture with k562 cells expressing BCMA, SLAMF7 or both. (A) Flow cytometry comparison of NFAT and NFκB reporters and CD69 MFI for CARtein-TPR cells co-cultured with k562 cells expressing BCMA, SLAMF7 or both surface proteins. Each dot represents an independent experimental triplicate. Statistical comparison between anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cells is indicated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). NFAT (B), NFκB (C) and CD69 (D) activation kinetics for anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cells upon co-culture with either, untransduced (UTD) or CARtein transduced k562 cells. Statistical analysis of Dual CARtein cells is shown for each time condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are average ± SEM of three different experimental replicates.

Figure 8

T cell activation signaling assay in CARtein Jurkat-TPR cells co-cultured with MM.1s cells. (A) SLAMF7 and BCMA expression in MM.1s cells. Schematic illustration of anti-BCMA or anti-SLAMF7 CARtein (B) or Dual CARtein (C) cells co-cultured with multiple myeloma target cells. (D) Representative flow cytometry analysis of NFAT and (E) NFκB activity and CD69 upregulation 24h after co-culture in a T:E ratio of 1:1. UTD, untransduced cells. JKT TPR, Jurkat-TPR cells.

Figure 9

Kinetics of anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cell activation upon MM.1s stimulation. (A) Flow cytometry-based statistical comparison of the mean fluorescence intensity (MFI) of NFAT and NFκB activity reporters and CD69 upregulation after co-culture with MM.1S cells at three different T:E ratios. Statistical analysis between T:E ratios is shown (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (B) NFAT, NFκB and CD69 activation kinetics for three T:E ratios of anti-BCMA, (C) anti-SLAMF7 and (D) Dual CARtein cells upon stimulation. Statistical analysis of CARtein cells in 1:1 ratio is indicated for each time condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are average ± SEM of three different experimental replicates. UTD, untransduced cells.

Figure 10

Comparison of CARtein-TPR cell activation upon stimulation with MM.1s or K562 cells expressing BCMA and SLAMF7. (A) Flow cytometry-based statistical comparison of the mean fluorescence intensity (MFI) of CD69 activation marker and (B) NFAT and NFκB reporter activity 24h after coculture at a target to effector ratio of 1:1. Statistical analysis between MM.1s and SLAMF7+/BCMA+ coculture is shown (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are ± SEM of three different experimental replicates. UTD, untransduced cells.