Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.     

Conjugate of ibrutinib with a TLR7 agonist suppresses melanoma progression and enhances antitumor immunity

The use of large molecules for immunotherapy has led to exciting developments in cancer treatment, such as the development of PD-1/PD-L1 antibodies. However, small molecule targeted therapies still lack effective immune-functional classes. Ideal anticancer drugs should simultaneously generate immune memory when killing cancer cells to prevent tumor relapse and metastasis. To this end, we carried out a rationally designed strategy to develop novel classes of small molecule compounds with bifunctional targeting and immunostimulatory abilities by conjugating targeting compounds with TLR7 agonists, generating immune-targeting conjugates (ImmunTacs). GY161, as a representative ImmunTac, was synthesized via chemical conjugation of ibrutinib with a TLR7 agonist. In vitro, GY161 stimulated the production of cytokines by mouse spleen lymphocytes, promoted the maturation of dendritic cells (DCs), and inhibited the growth and induced the apoptosis of B16 melanoma cells by regulating the c-Met/β-catenin pathway. In vivo, GY161 enhanced the frequency of CD8⁺ T cells in spleens and tumors, suppressed the growth of B16 melanoma cell-derived tumors and prolonged the survival time of mice. In summary, GY161 could prevent melanoma progression through direct tumor killing and by triggering specific immunity. These results strongly suggest that ImmunTacs are a reliable and promising strategy for developing small molecule immunogenic anticancer drugs.     

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function

The irreversible Bruton''s tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.     

Ibrutinib inhibits BTK-driven NF-κB p65 activity to overcome bortezomib-resistance in multiple myeloma

Multiple Myeloma (MM) is a haematologic malignancy characterized by the accumulation of clonal plasma cells in the bone marrow. Over the last 10–15 y the introduction of the proteasome-inhibitor bortezomib has improved MM prognosis, however relapse due to bortezomib-resistance is inevitable and the disease, at present, remains incurable. To model bortezomib-resistant MM we generated bortezomib-resistant MM cell lines (n = 4 ) and utilised primary malignant plasma cells from patients relapsing after bortezomib treatment (n = 6 ). We identified enhanced Bruton''s tyrosine kinase (BTK) activity in bortezomib-resistant MM cells and found that inhibition of BTK, either pharmacologically with ibrutinib (0.5 μM) or via lenti-viral miRNA-targeted BTK interference, re-sensitized previously bortezomib-resistant MM cells to further bortezomib therapy at a physiologically relevant concentration (5 nM). Further analysis of pro-survival signaling revealed a role for the NF-κB p65 subunit in MM bortezomib-resistance, thus a combination of BTK and NF-κB p65 inhibition, either pharmacologically or via further lenti-viral miRNA NF-κB p65 interference, also restored sensitivity to bortezomib, significantly reducing cell viability (37.5 ± 6 .9 %, ANOVA P ≤ 0 .001). Accordingly, we propose the clinical evaluation of a bortezomib/ibrutinib combination therapy, including in patients resistant to single-agent bortezomib.     

Ibrutinib modulates Aβ/tau pathology,neuroinflammation, and cognitive function in mouse models of Alzheimer's disease

We previously demonstrated that ibrutinib modulates LPS‐induced neuroinflammation in vitro and in vivo, but its effects on the pathology of Alzheimer''s disease (AD) and cognitive function have not been investigated. Here, we investigated the effects of ibrutinib in two mouse models of AD. In 5xFAD mice, ibrutinib injection significantly reduced Aβ plaque levels by promoting the non‐amyloidogenic pathway of APP cleavage, decreased Aβ‐induced neuroinflammatory responses, and significantly downregulated phosphorylation of tau by reducing levels of phosphorylated cyclin‐dependent kinase‐5 (p‐CDK5). Importantly, tau‐mediated neuroinflammation and tau phosphorylation were also alleviated by ibrutinib injection in PS19 mice. In 5xFAD mice, ibrutinib improved long‐term memory and dendritic spine number, whereas in PS19 mice, ibrutinib did not alter short‐ and long‐term memory but promoted dendritic spinogenesis. Interestingly, the induction of dendritic spinogenesis by ibrutinib was dependent on the phosphorylation of phosphoinositide 3‐kinase (PI3K). Overall, our results suggest that ibrutinib modulates AD‐associated pathology and cognitive function and may be a potential therapy for AD.     

Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.