B Cell Maturation Antigen (BCMA) has appeared as a promising target to use in CAR immunotherapy for multiple myeloma (MM) patients due to specific BCMA expression in plasma cells. We designed a 2nd generation CAR against BCMA adding 4-1BB as co-stimulatory domain. T cells were successfully transfected with our CAR against BCMA. CAR-T BCMA cells were functional in vitro against 3 different MM cell lines. Cytotoxicity assays were performed at ratios 1:1 and 0.5:1 of CAR-T: MM cell lines. All MM cells were eliminated completely at 72 hours. Moreover, CAR-T BCMA cells were functional in an in vivo NSG-mouse model with MM disease already established (Figure 1A).
Even published results are promising, high CAR-T cell doses have been needed to achieve clinical responses, which is associated with high toxicities. Moreover, loss of expression of the target antigen (BCMA in our study) appears in a proportion of patients with relapses, indicating that CAR-T cells will not recognize the tumor cell for any longer. Meanwhile, an ideal CAR treatment would be the one achieving permanent responses, with the lowest CAR cell dose possible, to avoid toxicities associated to the treatment.
We previously observed that when Natural Killer (NK) cells and MM cells become in contact there is concomitant transfer of NK cell proteins to the MM cell, and also MM proteins are transferred to NK cells. This transfer is performed in lipid structures, such as vesicles and lipid rafts. We hypothesized that after CAR-T and MM cell contact the same phenomenon could occur, with concomitant transfer of the CAR target antigen. This might explain why there is a loss of expression of the target antigen after CAR treatment. Therefore, we studied trafficking of BCMA between MM cells and CAR-T BCMA cells.
First, we analyzed BCMA expression in MM cells by confocal fluorescence microscopy and observed that BCMA is continuously being recycled and released in vesicles to the extracellular milieu. To analyze the trafficking of BCMA in MM cells after CAR-T BCMA exposure, we over-expressed BCMA fused to green fluorescent protein (GFP) in MM cells. MM cells over-expressing BCMA-GFP were co-cultured for 4 hours with CAR-T BCMA cells while time lapse in vitro confocal fluorescence microscopy was performed. BCMA transfer was observed to the extracellular milieu in vesicles and also to CAR-T BCMA cells. Moreover, flow cytometry analysis revealed that after 24h of co-culturing CAR-T BCMA cells with MM cells, a loss of BCMA surface expression in MM cells occurred, which was not observed with control non-transduced T cells (Figure 2B). Moreover, we observed that a proportion of CAR-T BCMA cells acquired BCMA expression.
To avoid this BCMA transfer we hypothesized that lipid synthesis inhibitors could be useful. Statins by inhibiting the Ac-CoA pathway, block cholesterol synthesis, and also they have an impact in the prenylation of proteins involved in protein degradation and recycling. Moreover, statins are anti-inflammatory agents and show anti-MM properties, suggesting their potential as co-adjuvants for CAR treatment. We tested and confirmed in vitro and in vivo the anti-MM properties of Fluvastatin. Moreover, 3h pre-treatment of MM cells with Fluvastatin before co-culturing with CAR-T BCMA cells decreased significantly loss of intracellular BCMA expression in MM cells (Figure 3C). Surface BCMA expression was the same, suggesting a decreased BCMA secretion to the extracellular milieu after statin treatment. Last, in a MM NSG-mouse model, pretreatment of mice with Fluvastatin (0.85mg/kg) for 4 days, before infusing CAR-T BCMA cells, did not impact negatively in the CAR-T BCMA cells activity. In conclusion, statins could be a good co-adjuvant to use in CAR immunotherapy treatment to prevent loss of BCMA antigen expression in the remaining MM cells after CAR treatment. Moreover, their anti-inflammatory properties could also improve side effects associated to this therapy.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.