Combined effects of exercise and immuno-chemotherapy treatments on tumor growth in MC38 colorectal cancer-bearing mice.

Acute exercise induces transient modifications in the tumor microenvironment and has been linked to reduced tumor growth along with increased infiltration of immune cells within the tumor in mouse models. In this study, we aimed to evaluate the impact of acute exercise before treatment administration on tumor growth in a mice model of MC38 colorectal cancer receiving an immune checkpoint inhibitor (ICI) and chemotherapy. Six-week-old mice injected with colorectal cancer cells (MC38) were randomized in 4 groups: control (CTRL), immuno-chemotherapy (TRT), exercise (EXE) and combined intervention (TRT/EXE). Both TRT and TRT-EXE received ICI: anti-PD1-1 (1 injection/week) and capecitabine + oxaliplatin (5 times a week) for 1 week (experimentation 1), 3 weeks (experimentation 2). TRT-EXE and EXE groups were submitted to 50 minutes of treadmill exercise before each treatment administration. Over the protocol duration, tumor size has been monitored daily. Tumor growth and microenvironment parameters were measured after the intervention on Day 7 (D7) and Day 16 (D16). From day 4 to day 7, tumor volumes decreased in the EXE/TRT group while remaining stable in the TRT group (p=0.0213). From day 7 until day 16 tumor volume decreased with no significant difference between TRT and TRT/EXE. At D7 the TRT/EXE group exhibited a higher total infiltrate T cell (p=0.0118) and CD8+ cytotoxic T cell (p=0.0031). At D16, tumor marker of apoptosis, vascular integrity and inflammation were not significantly different between TRT and TRT/EXE. Our main result was that acute exercise before immuno-chemotherapy administration significantly decreased early-phase tumor growth (D0 to D4). Additionally, exercise led to immune cell infiltration changes during the first week after exercise, while no significant molecular alterations in the tumor were observed 3 weeks after exercise.

A systemically administered detoxified TLR4 agonist displays potent antitumor activity and an acceptable tolerance profile in preclinical models.

Bacterial lipopolysaccharides (LPS) are potent innate immunostimulants targeting the Toll-like receptor 4 (TLR4), an attractive and validated target for immunostimulation in cancer therapy. Although LPS possess anti-tumor activity, toxicity issues prevent their systemic administration at effective doses in humans. We first demonstrated that LPS formulated in liposomes preserved a potent antitumor activity per se upon systemic administration in syngeneic models, and significantly enhance the antitumor activity of the anti-CD20 antibody rituximab in mice xenografted with the human RL lymphoma model. Liposomal encapsulation also allowed a 2-fold reduction in the induction of pro-inflammatory cytokines by LPS. Mice receiving an intravenous administration demonstrated a significant increase of neutrophils, monocytes and macrophages at the tumor site as well as an increase of macrophages in spleen. Further, we chemically detoxified LPS to obtain MP-LPS that was associated with a 200-fold decrease in the induction of proinflammatory cytokines. When encapsulated in a clinically approved liposomal formulation, toxicity, notably pyrogenicity (10-fold), was limited while the antitumor activity and immunoadjuvant effect were maintained. This improved tolerance profile of liposomal MP-LPS was associated with the preferential activation of the TLR4-TRIF pathway. Finally, in vitro studies demonstrated that stimulation with encapsulated MP-LPS reversed the polarization of M2 macrophages towards an M1 phenotype, and a phase 1 trial in healthy dogs validated its tolerance upon systemic administration up to very high doses (10µg/kg). Altogether, our results demonstrate the strong therapeutic potential of MPLPS formulated in liposomes as a systemically active anticancer agent, supporting its evaluation in patients with cancer.

In Vivo Syngeneic Tumor Models with Acquired Resistance to Anti-PD-1/PD-L1 Therapies.

Antibodies targeting PD-1 and PD-L1 have produced durable responses in a subset of patients with cancer. However, a majority of these patients will ultimately relapse due to acquired resistance. To explore the underlying mechanisms of this secondary resistance, we developed five syngeneic murine tumor variants with acquired resistance to anti-PD-1 and/or PD-L1 antibodies in vivo. Resistant in vivo models were obtained by serial treatment/reimplantation cycles of the MC38 colorectal, MB49 and MBT2 bladder, and RENCA kidney and TyrNras melanoma models. Tumor immune infiltrates were characterized for wild type and resistant tumors using spectral cytometry and their molecular alterations analyzed using RNA sequencing analyses. Alterations in the tumor immune microenvironment were strongly heterogeneous among resistant models, involving select lymphoid and/or myeloid subpopulations. Molecular alterations in resistant models included previously identified pathways as well as novel candidate genes found to be deregulated in several resistant models. Among these, Serpinf1, coding for pigment epithelial-derived factor (PEDF) was further explored in the MC38 and the MBT2 models. Overexpression of Serpinf1 induced resistance to anti-PD-1 antibodies in the MC38 model, whereas knockdown of Serpinf1 sensitized this model as well as the primarily resistant MBT2 model. Serpinf1 overexpression was associated with increased production of free fatty acids and reduced activation of CD8+ cells, while orlistat, a compound that reduces the production of free fatty acids, reversed resistance to anti-PD-1 therapy. Our results suggest that a panel of syngeneic resistant models constitutes a useful tool to model the heterogeneity of resistance mechanisms encountered in the clinic.

Proof of Concept: Protein Delivery into Human Erythrocytes Using Stable Cavitation.

Human erythrocytes represent candidates of choice as carriers for a wide range of drugs due to their unique biophysical and physiological properties. In this study, we used a sonoporation device generating and monitoring acoustic stable cavitation without any addition of contrast or nucleation agents. The device was evaluated for bovine serum albumin (BSA) delivery into human erythrocytes. After determining the adequate hematocrit percentage compatible with the generation of stable cavitation, we determined the optimal sonoporation conditions allowing BSA delivery while preserving erythrocyte integrity. Our results demonstrate that stable cavitation allows efficient delivery of proteins into human erythrocytes with limited lysis of these cells. In conclusion, our study allowed for the development of a stable and regulated cavitation program and the establishment of sonoporation conditions suitable for intracellular protein delivery while maintaining erythrocyte integrity. Additional investigations are needed to move from the proof of concept to a larger-scale application.

Impact of mouse model tumor implantation site on acquired resistance to anti-PD-1 immune checkpoint therapy.

Introduction: The use of tumor subcutaneous (SC) implantations rather than orthotopic sites is likely to induce a significant bias, in particular, in the field of immunotherapy. Methods: In this study, we developed and characterized MC38 models, implanted subcutaneously and orthotopically, which were either sensitive or rendered resistant to anti-PD1 therapy. We characterized the tumor immune infiltrate by flow cytometry at baseline and after treatment. Results and Discussion: Our results demonstrate several differences between SC and orthotopic models at basal state, which tend to become similar after therapy. These results emphasize the need to take into account tumor implantation sites when performing preclinical studies with immunotherapeutic agents.

Exatecan Antibody Drug Conjugates Based on a Hydrophilic Polysarcosine Drug-Linker Platform.

We herein report the development and evaluation of a novel HER2-targeting antibody-drug conjugate (ADC) based on the topoisomerase I inhibitor payload exatecan, using our hydrophilic monodisperse polysarcosine (PSAR) drug-linker platform (PSARlink). In vitro and in vivo experiments were conducted in breast and gastric cancer models to characterize this original ADC and gain insight about the drug-linker structure-activity relationship. The inclusion of the PSAR hydrophobicity masking entity efficiently reduced the overall hydrophobicity of the conjugate and yielded an ADC sharing the same pharmacokinetic profile as the unconjugated antibody despite the high drug-load of the camptothecin-derived payload (drug-antibody ratio of 8). Tra-Exa-PSAR10 demonstrated strong anti-tumor activity at 1 mg/kg in an NCI-N87 xenograft model, outperforming the FDA-approved ADC DS-8201a (Enhertu), while being well tolerated in mice at a dose of 100 mg/kg. In vitro experiments showed that this exatecan-based ADC demonstrated higher bystander killing effect than DS-8201a and overcame resistance to T-DM1 (Kadcyla) in preclinical HER2+ breast and esophageal models, suggesting potential activity in heterogeneous and resistant tumors. In summary, the polysarcosine-based hydrophobicity masking approach allowsfor the generation of highly conjugated exatecan-based ADCs having excellent physicochemical properties, an improved pharmacokinetic profile, and potent in vivo anti-tumor activity.

Calcium Channel Blockers Impair the Antitumor Activity of Anti-CD20 Monoclonal Antibodies by Blocking EGR-1 Induction.

Direct cell death induction, in addition to immune-effector cell-mediated mechanisms, is one of the key mechanisms of action of anti-CD20 antibodies, and yet the signaling pathways implicated remain poorly investigated. Here we show that the transcription factor EGR-1 is rapidly induced by anti-CD20 antibodies and is a key mediator for CD20-induced cell death. EGR-1 induction results from an increased calcium influx induced by anti-CD20 antibodies. We show that both rituximab and obinutuzumab induce calcium influx, albeit through different mechanisms, and this influx is crucial for cell death induction. Inhibition of the calcium flux with calcium channel blockers (CCB) abolished EGR-1 induction and impaired the efficacy of anti-CD20 antibodies in preclinical in vitro and in vivo models. Finally, we investigated the impact of CCBs in patients treated with anti-CD20 antibodies included in the clinical trials GOYA and REMARC, and found that patients simultaneously receiving CCBs and anti-CD20 therapy have a shorter progression-free survival and overall survival. These results reveal EGR-1 as a key mediator of the direct cytotoxic activity of anti-CD20 antibodies and provide a rationale to evaluate EGR-1 expression as a new biomarker to predict response to anti-CD20 treatment. In addition, our findings show that calcium influx is required for anti-CD20-mediated tumor cell death and suggest that simultaneous administration of calcium channel blocking agents could be deleterious in patients receiving anti-CD20-based immunotherapy.

Characterization of T-DM1-resistant breast cancer cells.

The development of targeted therapies has drastically improved the outcome of patients with different types of cancer. T-DM1 (trastuzumab-emtansine) is an antibody-drug conjugate used for the treatment of HER2-positive breast cancer combining the FDA approved mAb (monoclonal antibody) trastuzumab and the microtubule cytotoxic agent DM1 (emtansine). Despite clinical successes achieved by targeted therapies, a large number of patients develop resistance during treatment. To explore mechanisms of resistance to T-DM1, the MDA-MB-361 HER2-positive breast cancer cell line was exposed in vitro to T-DM1 in the absence or presence of ciclosporin A. Previously reported mechanisms of resistance such as trastuzumab-binding alterations, MDR1 upregulation, and SLC46A3 downregulation were not observed in these models. Despite a decrease in HER2 expression at the cell surface, both resistant cell lines remained sensitive to HER2 targeted therapies such as mAbs and tyrosine kinase inhibitors. In addition, sensitivity to DNA damaging agents and topoisomerase inhibitors were unchanged. Conversely resistance to anti-tubulin agents increased. Resistant cells displayed a decreased content of polymerized tubulin and a decreased content of ßIII tubulin although the downregulation of ßIII tubulin by siRNA in the parental cell line did not modified the sensitivity to T-DM1. Both cell lines resistant to T-DM1 also presented giant aneuploid cells. Several SLC (solute carrier) transporters were found to be differentially expressed in the resistant cells in comparison to parental cells. These results suggest that some characteristics such as increased baseline aneuploidy and altered intracellular drug trafficking might be involved in resistance to T-DM1.

A heavy metal baseline score predicts outcome in acute myeloid leukemia.

Despite abundant epidemiological data linking metals to leukemia and other cancers, baseline values of toxic and essential metals in patients with leukemia and the clinical impact of these metals remain unknown. Thus, we sought to quantify metal values in untreated patients with acute myeloid leukemia (AML) and controls and determine the impact of metal values on AML patients' survival. Serum samples from patients with untreated AML and controls at Hospices Civils de Lyon were analyzed and compared for trace metals and copper isotopic abundance ratios with inductively coupled plasma mass spectrometry. Survival analysis was performed as a function of metal values, and a multi-metal score was developed for patients with AML. Serum samples were collected from 67 patients with untreated AML and 94 controls. Most patients had intermediate-risk cytogenetics (63.1%) without FLT3 internal tandem duplication mutations (75.6%) or NPM1 mutations (68.1%). Most metal values differed significantly between AML and control groups. Patients with lower magnesium and higher cadmium values had the worst survival rates, with only 36% surviving at 6 months (P = .001). The adverse prognostic effect of this combination was maintained on multivariate analysis. Based on this, we developed a novel metal score, which accounts for multiple relative abnormalities in the values of five toxic and five essential metals. Patients with a higher metal score had significantly worse survival, which was maintained on multivariate analysis (P = .03). This baseline metal scoring system was also prognostic when we applied it to a separate population of front-line AML patients.

The Antitumor Activity of Combinations of Cytotoxic Chemotherapy and Immune Checkpoint Inhibitors Is Model-Dependent.

In spite of impressive response rates in multiple cancer types, immune checkpoint inhibitors (ICIs) are active in only a minority of patients. Alternative strategies currently aim to combine immunotherapies with conventional agents such as cytotoxic chemotherapies. Here, we performed a study of PD-1 or PDL-1 blockade in combination with reference chemotherapies in four fully immunocompetent mouse models of cancer. We analyzed both the in vivo antitumor response, and the tumor immune infiltrate 4 days after the first treatment. in vivo tumor growth experiments revealed variable responsiveness to ICIs between models. We observed enhanced antitumor effects of the combination of immunotherapy with chemotherapy in the MC38 colon and MB49 bladder models, a lack of response in the 4T1 breast model, and an inhibition of ICIs activity in the MBT-2 bladder model. Flow cytometry analysis of tumor samples showed significant differences in all models between untreated and treated mice. At baseline, all the tumor models studied were predominantly infiltrated with cells harboring an immunosuppressive phenotype. Early alterations of the tumor immune infiltrate after treatment were found to be highly variable. We found that the balance between effector cells and immunosuppressive cells in the tumor microenvironment could be altered with some treatment combinations, but this effect was not always correlated with an impact on in vivo tumor growth. These results show that the combination of cytotoxic chemotherapy with ICIs may result in enhanced, similar or reduced antitumor activity, in a model- and regimen-dependent fashion. The present investigations should help to select appropriate combination regimens for ICIs.

Piperidinyl-embeded chalcones possessing anti PI3Kδ inhibitory properties exhibit anti-atopic properties in preclinical models.

Phosphatidylinositide 3-kinases (PI3Ks) are widely expressed enzymes involved in membrane signalization pathways. Attempts to administer inhibitors with broad activity against different isoforms have failed due to toxicity. Conversely the PI3Kδ isoform is much more selectively expressed, enabling therapeutic targeting of this isoform. Of particular interest PI3Kδ is expressed in human basophils and its inhibition has been shown to reduce anti-IgE induced basophil degranulation, suggesting that PI3Kδ inhibitors could be useful as anti-allergy drugs. Herein, we report for the first time the activity of compounds derived from chalcone scaffolds as inhibitors of normal human basophil degranulation and identified the most active compound with anti-PI3Kδ properties that was investigated in preclinical models. Compound 18, namely 1-[2-hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-4-yl)phenyl]-3-(2,4,6-trimethoxyphenyl)-prop-2-en-1-one, was found to inhibit normal human basophil degranulation in a dose-dependent manner. In a murine model of ovalbumin-induced asthma, compound 18 was shown to reduce expiratory pressure while its impact on the inflammatory infiltrate in alveolar lavage and total lung was dependent on the route of administration. In a DNFB-induced model of atopic dermatitis compound 18 administered systemically proved to be as potent as topical betamethasone. These results support the anti-atopic and allergic properties of the title compound and warrant further clinical development.

Unexpected Growth-Promoting Effect of Oxaliplatin in Excision Repair Cross-Complementation Group 1 Transfected Human Colon Cancer Cells.

The nucleotide excision repair protein excision repair cross-complementation group 1 (ERCC1) has been repeatedly shown to be involved in the sensitivity of cancer cells to platinum derivatives. In order to better understand this process, we transfected HCT-116 cells with a plasmid encoding ERCC1 and studied their in vitro and in vivo behaviour. No main differences were observed for sensitivity to platinum drugs, DNA repair capacity and clonogenicity in vitro. However, -ERCC1-transfected HCT-116 cells showed paradoxical behaviour in vivo with increased growth in mice treated with oxaliplatin as compared to untreated mice. The Trop2 protein was identified as being potentially involved in the underlying mechanism for these observations, as it was overexpressed in transfected cells. Our results suggest complex regulation of signalling in cancer cells exposed to cancer drugs.

Esophageal cancer cells resistant to T-DM1 display alterations in cell adhesion and the prostaglandin pathway.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate that specifically targets HER2 thanks to its antibody component trastuzumab. In spite of responses to this novel agent, acquired resistance to treatment remains a major obstacle. Prolonged in vitro exposure of the gastroesophageal junction cancer cell line OE-19 to T-DM1, in the absence or presence of ciclosporin A resulted in the selection of two resistant cell lines to T-DM1. T-DM1-resistant cells presented an increased expression of adhesion genes, altered spreading and higher sensitivity to anoikis than parental cells. A resistant cell line showed decreased adhesion strength, increased migration speed and increased sensitivity to RhoA inhibition. Genes involved in the prostaglandin pathway were deregulated in resistant models. Addition of prostaglandin E2 to T-DM1 partially restored its cytotoxic activity in resistant models. This work demonstrates that T-DM1-resistance may be associated with alterations of cell adhesion and the prostaglandin pathway, which might constitute novel therapeutic targets.

Neutrophils protect lymphoma cells against cytotoxic and targeted therapies through CD11b/ICAM-1 binding.

Innate immune cells constitute a substantial proportion of the cells within the tumor microenvironment. Besides the contribution of the microenvironment to tumor proliferation and survival, there is direct evidence that interactions between tumor cells and their microenvironment alter sensitivity to anti-cancer agents. Neutrophils, a key player in the innate immune system, have been less studied than many other immune cells regarding their impact on cancer cell response to anti-cancer agents. In our 2D and 3D coculture systems, human neutrophils and differentiated HL60 cells attenuated the sensitivity of various lymphoma cell lines to several anti-cancer agents, including targeted therapies. Neutrophil-induced protection was dependent on cell-cell interaction between CD11b and ICAM-1 expressed by neutrophils and B cells, respectively and was shown to be Mcl-1-dependent. The protective effect of neutrophils was validated in vivo using immune-compromised mice inoculated with human NHL with our without neutrophils then followed by treatment with chemotherapy. Similar findings were made on primary cells purified from patients with chronic lymphocytic leukemia, treated with fludarabine or targeted agents in the presence of autologous neutrophils. In a clinical study, patients with non-Hodgkin's lymphoma with increased neutrophil counts displayed a reduced response rate to therapy. These findings reveal a novel protective mechanism of neoplastic B cells involving innate immune cells which could be pharmacologically targeted to enhance the antitumor effect of therapy.

Deoxycholic acid derivatives as inhibitors of P-glycoprotein-mediated multidrug efflux.

Deoxycholic acid derivatives were designed as P-glycoprotein (Pgp, ABCB1) inhibitors. Thus the synthesis and the biological activity of methyl deoxycholate derivatives 5-10 and their ether analogs 15-20 have been reported. The potency of these compounds to modulate Pgp-mediated MDR was evaluated through daunorubicin accumulation and potentiation of doxorubicin cytotoxicity in K562/R7 multidrug resistant cells overexpressing Pgp. In parallel, their intrinsic toxicity was appreciated on K562 sensitive cells. Methyl 12α-[(2R or 2S) tetrahydro-2H-pyran-2-yloxy]-3-oxo-5ß-cholan-24-oate 9b has shown a good efficiency as a Pgp inhibitor and a low intrinsic toxicity. Therefore, this derivative constitutes a new lead compound which can be used as a starting point to improve the design of non-toxic Pgp modulators.

Pegfilgrastim Enhances the Antitumor Effect of Therapeutic Monoclonal Antibodies.

Therapeutic mAbs exert antitumor activity through various mechanisms, including apoptotic signalization, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP). G-CSF and GM-CSF have been reported to increase the activity of antibodies in preclinical models and in clinical trials. To determine the potential role of pegfilgrastim as an enhancer of anticancer antibodies, we performed a comparative study of filgrastim and pegfilgrastim. We found that pegfilgrastim was significantly more potent than filgrastim in murine xenograft models treated with mAbs. This was observed with rituximab in CD20(+) models and with trastuzumab in HER2(+) models. Stimulation with pegfilgrastim was associated with significant enhancement of leukocyte content in spleen as well as mobilization of activated monocytes/granulocytes from the spleen to the tumor bed. These results suggest that pegfilgrastim could constitute a potent adjuvant for immunotherapy with mAbs possessing ADCC/ADCP properties. Mol Cancer Ther; 15(6); 1238-47. ©2016 AACR.

Adipose cells promote resistance of breast cancer cells to trastuzumab-mediated antibody-dependent cellular cytotoxicity.

INTRODUCTION: Trastuzumab has been used in the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer, but its efficacy is limited by de novo or acquired resistance. Although many mechanisms have been proposed to explain resistance to trastuzumab, little is known concerning the role of the tumor microenvironment. Given the importance of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor effect of trastuzumab and the abundance of adipose tissue in the breast, we investigated the impact of adipocytes on ADCC. METHODS: We set up a coculture system to study the effect of adipocytes on ADCC in vitro. The results were validated in vivo in a mouse xenograft model. RESULTS: We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC in HER2-expressing breast cancer cells via the secretion of soluble factors. The inhibition of ADCC was not due to titration or degradation of the antibody. We found that adipose cells decreased the secretion of interferon-γ by natural killer cells, but did not alter natural killer cells' cytotoxicity. Preincubation of breast cancer cells with the conditioned medium derived from adipocytes reduced the sensitivity of cancer cells to ADCC. Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium. Importantly, breast tumors grafted next to lipomas displayed resistance to trastuzumab in mouse xenograft models. CONCLUSIONS: Collectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte-cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.

Synthesis of new steroidal inhibitors of P-glycoprotein-mediated multidrug resistance and biological evaluation on K562/R7 erythroleukemia cells.

A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/ß-pregnane-3,20-dione or 5ß-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/ß-pregnane-3,20-dione, among which the 5ß-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 µM versus 1.2 and 10.6 µM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5ß-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 µM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

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.