The use of CD44v6-CAR T cells in combination with PD-1/PD-L1 and PI3K/AKT pathway inhibitors as therapeutic strategies for advanced cancer

BackgroundDespite the recent advances in cancer care standards, a notable number of patients fail to respond to first-line standard treatments or become refractory after an initial partial response. These phenomena are attributed to the presence of a subpopulation of cancer cells named cancer stem cells (CSCs), which are responsible for tumor initiation, chemoresistance, and spreading.CSCs isolated from several tissues, including colorectal (CRC) and breast (BC) cancer, express the stemness marker CD44v6, which is a crucial molecule required to boost cell metastatic potential. In the last few years, cancer adoptive immunotherapy based on the use of chimeric-antigen receptor (CAR) T cells has been developed as a ground-breaking therapy to successfully tackle cancer by enforcing patients’ own immune responses. Aim of the studyThis research aimed at establishing an effective procedure for the generation of CD44v6-CAR T cells able to selectively eliminate CD44v6-positive CSCs derived from CRC and BC patients. Moreover, potential strategies to strengthen CD44v6-CAR T cells performance have been investigated.DesignThe initial phase of the study focused on the procedures for the isolation, enrichment, and expansion of T cells obtained from the peripheral blood of healthy donors. Subsequently, T cells were engineered with a second-generation CAR lentiviral vector containing i) an extracellular antigen-recognizing single-chain variable fragment (scFv) targeting CD44v6, ii) a hinge transmembrane domain, iii) an intracellular complex that includes CD3 signalling and CD28 co-stimulatory domains, and iv) a GFP coding sequence.We collected primary sphere cell cultures from colorectal cancer (CRC) and breast cancer (BC) specimens. To examine the potential of CD44v6-CAR T cell treatments against CR-CSCs and BCSCs cytotoxic assays have been performed.Moreover, to further optimize the employment of CD44v6-CAR T cells, we investigated tumor microenvironment (TME) influence on CAR T cells performance. RNA-seq analysis has been used to identify targetable biomarkers that could improve CD44v6-CAR T cells efficacy. ResultsHere, we showed an optimized procedure to isolate and expand T cells engineered with CD44v6-CAR with a high transduction efficiency (45±38,5%). In vitro co-culture experiments demonstrated that CD44v6-CAR T cells effectively eradicated CR-CSCs and BCSCs bearing a high expression of CD44v6. Moreover, our preliminary data demonstrated that tumor microenvironment (TME) could foster immune escape, influencing the effectiveness of immunotherapy. In particular, a whole transcriptomic analysis revealed a positive regulation of the immune suppressor PD-L1, on CSC, following the exposure to adipose stem cells (ASCs) resident TME components. Likewise, we have shown that TME sustains the ability of CD44v6-positive CSCs to proliferate and metastasize through the PI3K/AKT pathway. Contextually, it has been proved that the PI3K/AKT pathway limits CAR T cell expansion and persistence. Hence, combined inhibition of PD-L1 and PI3K/AKT pathway could be a feasible strategy to normalize the dysregulated TME and reinforce CD44v6-CAR T cells efficacy.ConclusionsThis data supports the use of CD44v6-CAR T cells as a potential treatment to prevent CSCs proliferation and seeding at distant organs. Moreover, these data provide a rationale for strengthening the cytotoxic effect and persistence of CD44v6-CAR T cells by designing combinatorial therapies including PD-L1 and PI3K/AKT inhibitors.