Driving Engineered CARgos Against Multiple Myeloma
CAR-T therapy has revolutionized the treatment of hematological malignancies, yet many patients still relapse due to tumor resistance mechanisms. In multiple myeloma (MM), we have found that overexpression of the anti-apoptotic protein MCL-1 impairs anti-BCMA (B-cell maturation antigen) CAR-T cell-mediated killing, and therefore helps the tumors escape therapy. Specific inhibitors of MCL-1 may overcome this resistance and support CAR-T action, but their use is limited as they exert cardiac toxicity. To overcome this, we sought a way to inhibit MCL-1 in a targeted way by locally delivering the BH3-only protein NOXA, the endogenous antagonist of MCL-1.
We first assessed the expression of MCL-1 in MM cell lines and patient samples following co-culture with anti-BCMA. In order to specifically target MCL-1 in tumors cells, we generated anti-BCMA CAR-T cells overexpressing granzyme B bound to NOXA as a cargo through a cathepsin D-cleavable linker. This way, lytic granules containing both granzyme B and NOXA are released upon immune synapse formation. Generated T cells, namely (NOXA)-CARgo-T cells, were tested for cytotoxic activity in vitro against cell lines and primary samples. They were then further assessed in vivo in NSG mice bearing MM xenograft of luciferase-expressing cell lines. Off-tumor safety of NOXA was evaluated in co-culture experiments of engineered CAR-T cells and primary bone marrow stromal cells or IPSC-derived cardiomyocytes.
We found that MM cells surviving exposure to standard anti-BCMA CAR T-cells displayed elevated MCL-1 levels, suggesting selection for apoptosis-resistant clones. MCL-1 inhibition restored sensitivity to CAR T-mediated killing, confirming the role of MCL-1 in resistance. Using the fluorescent protein mScarlet as a cargo, we confirmed via fluorescent microscopy the correct expression and localization of Granzyme B-cargo in LAMP-1 positive granules, as well as its delivery to the target cells. NOXA-CARgo-T cells exhibited enhanced cytotoxicity and higher apoptosis against both MM cell lines and primary samples compared to cells expressing an inactive mutant of NOXA. In vivo, NOXA-CARgo-T cells demonstrated a stronger and more durable tumor suppression in MM xenograft. Moreover, our generated NOXA-CARgo-T cells did not affect the viability of non-target bone marrow stromal cells or cardiomyocytes.
We demonstrate that arming CAR T cells with NOXA as a pro-apoptotic payload can overcome apoptosis resistance in MM. By locally delivering NOXA to MM cells, we successfully enhanced killing efficacy without systemic toxicity. This modular strategy paves the way to testing additional CARgo payloads that could further enhance MM treatment. It also provides a framework to explore other cell death modalities beyond apoptosis, such as immunogenic cell death. To this end, we are developing biosensor-expressing MM cell lines to better monitor the underlying mechanisms of CARgo-T cell induced tumor cell death.