BTK Inhibition Prevents the Induction of Myeloid-Derived Suppressor Cells in Malignancy and Improves CART Function

The addition of the BTK inhibitor ibrutinib to CART therapy increased complete response rate from 28% (n = 32) to 53% (n = 19) in patients with chronic lymphocytic leukemia (CLL) in two sequential studies at our institution (J Clin Oncol. 2020 Sep 1:2862-71; Blood Advances. 2022 Nov:4774-85). This observation was hypothesized to stem from direct inhibition of the related kinase ITK in patient T cells and/or from reduction of CLL burden with secondary effects on T cell quality (Blood. 2016 Mar:1117-27). However, BTK is also expressed in myeloid cells, and ibrutinib has been shown to deplete myeloid derived suppressor cells (MDSCs) in murine tumor models (Cancer Res. 2016 Apr:2125-36). Here we sought to systematically define the mechanism(s) by which ibrutinib improves CART function.

We first compared the function of residual CART19 infusion products biobanked from a clinical trial of monotherapy (NCT01747486) to one in which patients also received ibrutinib starting at least six months prior to infusion (NCT02640209). We used a leukemia cell line (NALM6) as a universal target. There was no difference in killing (p = 0.76, n = 10) or antigen-specific proliferation (p = 0.74, n = 18), suggesting that ibrutinib treatment did not meaningfully affect CART19 quality despite substantial differences in patient responses.

We next evaluated patient myeloid cells in peripheral blood (PB) samples taken the day prior to infusion (D-1). Patients not receiving ibrutinib (n=9) had a median of 144 immunophenotypic M-MDSCs per uL of PB (range 3-241 cells/uL) compared to a median of 49/uL (range 11-110 cells/uL) among patients who were (n = 10, p = 0.024). We then tested the ability of these myeloid cells to inhibit autologous CART19 cells. The addition of CD14⁺ myeloid cells from ibrutinib-untreated patients to CART19 and NALM6 cell co-culture reduced the mean percent proliferating CART19 cells from 92.4% to 64.2% (p = 0.0097), while CD14⁺ cells from ibrutinib treated patients did not impair T cell proliferation (95.6% vs 97.0%; p = 0.49).

MDSCs are thought to form in response to soluble factors elaborated in the context of systemic malignancy. We thus hypothesized that patient serum could induce MDSC formation. Indeed, the presence of healthy donor (HD) CD14⁺ exposed for five days to serum from ibrutinib-untreated patients reduced mean antigen stimulated CART19 cell proliferation from 93.2% to 59.7% (p = 0.01). This inhibition was reversed if the serum was supplemented with 1 uM ibrutinib (mean 95.3% CART19 cells proliferating; p = 0.002). In contrast, the serum of patients receiving ibrutinib did not convert HD CD14⁺ cells to MDSC as the presence of these cells in co-culture did not inhibit CART19 proliferation (p = 0.99). This sera is assumed to contain ibrutinib, and we also compared its cytokine prolife to that of ibrutinib-untreated patients. Ibrutinib-treated patient serum had lower levels of the MDSC associated cytokines IL-10 (p = 0.007), IL-13 (p = 0.0008), GM-CSF (p < 0.0001), and IL-4 (p = 0.03), but higher levels of the Th1 secreted cytokines TNFα (p = 0.0009), IL-2 (p = 0.0045), and IFNγ (p < 0.0001).

Since ibrutinib inhibits several kinases, we tested a more selective BTK inhibitor (acalabrutinib) and again found that immunophenotypic and functionally defined MDSC formation was prevented (p < 0.0001 for both analyses).

To further validate the role of BTK in MDSC development, we deleted BTK in HD CD14⁺ cells. BTK-sufficient HD CD14⁺ cells exposed to IL-6 and GM-CSF over 4 days developed into CD33⁺HLA-DR^low MDSC capable of potently suppressing CART19 proliferation, while BTK-deficient HD CD14 did not (p = 0.04 for phenotypic comparison, p = 0.002 for functional comparison).

Finally, we explored if the effect of ibrutinib on MDSC formation was generalizable beyond CLL. Exposure to serum samples collected on D-1 from four multiple myeloma or three pancreatic cancer patients treated on institutional CART trials polarized HD CD14⁺ cells to potently suppressive MDSC (p = 0.012 and p = 0.008 respectively), and this suppressive effect was abolished in the presence of ibrutinib (p = 0.98 and p = 0.99 respectively).

These data suggest that ibrutinib improves CART19 therapy in CLL not by generating functionally superior CART cells per se, but by preventing the formation of MDSC, thus removing a critical counter-regulatory break on CART function. These results provide a mechanism by which ibrutinib can enhance CART therapy in CLL and beyond.