Intein-based modular assembly of multi-receptor CAR T cells Free

Background: Chimeric antigen receptor (CAR) T cells demonstrate strong antitumor activity. However, therapeutic success is often limited by heterogeneous or insufficient tumor-associated antigen expression preventing tumor cell recognition, suboptimal or dysfunctional CAR T cell signaling preventing tumor elimination, and on-target off-tumor activity causing toxicity. Expressing multiple CARs or combining CARs with chimeric costimulatory receptors (CCRs), which provide additional costimulation, can overcome heterogeneous or weak antigen expression. Substituting standard CD3ζ-based CAR signaling domains with kinase-based alternatives such as Zap70 may reduce dysfunctional tonic signaling and improve T cell activity. Drug-regulated CARs can further limit off-tumor toxicity. However, engineering complexity and vector packaging constraints pose challenges for combining multiple strategies within a single cell therapy product. To overcome these limitations, we developed a Trans-splicing Intein-Generated Extein-Recombination (TIGER) system, which directs post-translational, combinatorial assembly of multiple co-expressed drug-regulated chimeric receptors.

Methods: We used orthogonal trans-splicing inteins to co-express multiple chimeric receptors in T cells by combining shared and reused modular receptor components, which reduced the amount of DNA required to encode multiple receptors. To facilitate increased transgene delivery to T cells, we combined intein-dependent endoplasmic reticulum retention of affinity-tagged transmembrane proteins with multi-vector transduction and immunomagnetic selection. This approach enabled purification of T cells simultaneously incorporating up to four vectors. We generated drug-regulated multi-CAR/CCR T cells that assembled multiple co-expressed chimeric receptors containing standard CAR signaling domains (e.g., CD28-CD3ζ, 4-1BB-CD3ζ) or kinase-based domains (e.g., Zap70), CCR signaling domains (e.g., 4-1BB, CD28BB), and hepatitis C virus NS3-protease-based drug regulation domains. We confirmed receptor assembly and CAR T cell function by flow cytometry, mass spectrometry, and live-cell imaging of CAR T cell proliferation and tumor killing in vitro.

Results: We co-expressed an array of two drug-regulated CARs and two CCRs using a DNA template in which each modular domain was encoded only once. Successful receptor assembly was confirmed by mass spectrometry. Intein-assembled receptor arrays incorporating CD19 and CD20 binders, CD28ζ and 4-1BB signaling domains, and an NS3-protease drug-regulation domain directed drug-dependent T cell killing of CD19⁺ or CD20⁺ leukemia cells in vitro. Inclusion of 4-1BB CCR enhanced T cell proliferation, which was associated with enhanced overall tumor killing and T cell persistence following repetitive target stimulations. Despite the large size of an NS3-Zap70 drug-regulated signaling domain (~2 kb), we successfully generated a functional drug-regulated dual-CAR dual-CCR array, saving over 3 kb of packaging capacity versus conventional receptor encoding and achieving 1.7× compression. Finally, by combining variable numbers of CD3ζ-based, costimulatory, and non-signaling delta domains, we observed graded T cell activation with increasing signaling domain usage as measured by high-dimensional flow cytometry.

Conclusions: We present an intein-based post-translational combinatorial receptor assembly system to facilitate the co-expression of ensembles of receptors. This system required less DNA packaging space compared with conventional encoding approaches and simplified the engineering of multi-antigen-specific CAR T cells with large kinase-based signaling domains and modular drug-regulated functionality. Combining CD3ζ or Zap70-based signaling domains with CCR costimulatory domains through the intein-based system enhanced T cell functionality and persistence after multiple rounds of antigen stimulation. By varying stoichiometric ratios of stimulatory and co-stimulatory domains, we observed gradations in antigen-stimulated CAR T cell activation. These results suggest that co-expression of multiple receptors activating different signal transduction pathways can be varied to optimize T cell function. Additionally, our intein-based system provides a modular platform for evaluation and screening of multi-antigen targeting CAR T cells with diverse antigen binding and intracellular signaling domain combinations.