Metabolic reprogramming of Trimeric APRIL-CAR-engineered Natural Killer (NK) cells to enhance tumor-cell recognition, in vivo persistence and anti-tumor potency in Multiple myeloma

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Chimeric-antigen receptor (CAR)-based cellular immunotherapy has augmented the treatment armamentarium for aggressive B cell lymphomas and acute lymphoid leukemia with autologous CAR-T products now routinely applied in the clinic. However, significant logistic obstacles of generating autologous cell products make this treatment modality cumbersome and available only to a selected group of patients. Allogeneic cord blood derived natural killer (NK) cells promise to overcome these hurdles and can be administered without the need for full HLA matching as an off-the-shelve product.Multiple myeloma is an incurable clonal plasma cell malignancy with a median overall survival of 6 years despite adoption of novel substances such as proteasome inhibitors and immunomodulatory drugs. While the outlook of BCMA-directed autologous CAR-T therapies promises to further improve clinical outcomes, eventual relapse due to the emergence of BCMA-negative disease and limited in vivo persistence remain a challenge.Multi-specific targeting, in which CAR-modified immune cells are engineered to recognize multiple tumor-associated antigens, promises to overcome antigen-loss driven immune escape. Natural ligand CARs which rely on physiologically occurring high affinity ligands to selectively bind to their receptor counterparts allow to simultaneously target multiple tumor-associated antigens. In multiple myeloma, APRIL is such a natural ligand and enables CAR-NK cells to engage with the plasma cell specific antigens TACI and BCMA. In the proposed project we plan to engineer a novel dual-specific trimeric APRIL-CAR-NK construct which replicates APRIL's natural trimeric conformation using a multicistronic retroviral vector platform to enhance plasma cell binding affinity. Specific plasma cell lysis will be investigated using flow-cytometry based cytotoxicity assays and augmented by visualization of immunological synapse formation by confocal microscopy.Addressing the limited in vivo persistence of current generation CAR-T therapies, we plan to modulate the immunometabolomic signature and increase CAR-NK cellular fitness. Immune responses are crucially dependent on intracellular nutrient levels and MYC signaling in particular is as a key regulator of NK cell metabolic configuration. We therefore hypothesize that modulating the activity of MYC signaling can render CAR-NK cells more metabolically robust and enhance in vivo persistence. To address this idea, we will fine-tune intracellular MYC levels by targeting their post-translational regulatory networks, specifically the SUMO-Ubiquitin crosstalk. Reprogrammed CAR-NK cells will be characterized in-depth using a multi-OMICS approach relying on mass-spectrometry-based metabolomics and single cell transcriptomic profiling to elucidate their altered metabolomic signatures. Functionally, reprogrammed CAR-NK cells will be validated in vivo using a BCMA-negative multiple myeloma mouse model.