Inhibition of Bruton's Tyrosine Kinase (BTK) in the treatment of B-cell malignancies has been an exemplary story of functional understanding of disease pathogenesis translating to superior survival rates (1) (2) (3) (4) . Moreover, inhibition of BTK is currently also being evaluated as a therapeutic strategy for patients with severe COVID-19 disease (5, 6) . BTK is localized in proximity of the B-cell receptor (BCR), forming a signalosome complex upon BCR activation with other BCR associated kinases (BAK); LYN, SYK, and PI3K (7, 8) . The first generation BTK inhibitor (BTKi) ibrutinib covalently binds its target, which leads to prolonged lymphocytosis via reduced malignant B-cell homing capacity and chemokine signaling (9) . However, ibrutinib also has high affinity to other Tec kinases, leading to the development of second generation BTKis, such as tirabrutinib and acalabrutinib, which are highly selective for BTK (10, 11) .
After initial success of ibrutinib in early phase clinical trials as a single agent in the relapse/refractory setting, clinical trials were quickly initiated in combination with other frontline agents, namely monoclonal antibodies targeting CD20 (anti-CD20; such as rituximab) (12) (13) (14) (15) . The most recent phase 3 trial in previously untreated chronic lymphocytic leukemia (CLL) offers superior survival in comparison to the chemo-immunotherapeutic (CIT) regimen fludarabine cyclophosphamide and rituximab (FCR) (12) . Rituximab elicits its therapeutic effect via direct cell killing, complement activation, and Fc-mediated antibody dependent cellular cytotoxicity (ADCC) (16) . In addition, CIT has been previously shown to induce an acute secretory phenotype leading to increased macrophage mediated antibody dependent cellular phagocytosis (ADCP), in a humanized mouse model of B-cell lymphoma (17) (18) (19) . Therefore, it is important to evaluate the synergistic interaction of BTK inhibition and monoclonal antibody treatment in the context of the tumor microenvironment (TME). 4 Pre-clinical studies assessing the synergistic interaction between ibrutinib and monoclonal antibodies have provided conflicting results, with a number of studies reporting that ibrutinib not only has no impact on antibody-mediated effects, rather a negative effect specifically on NK-cell mediated effects of the antibody (20) (21) (22) (23) .
Down-regulation of CD20 on CLL cells by ibrutinib has additionally been described in co-culture systems and may be interpreted as a potential antagonistic mechanism (24, 25) . However, combination therapy of ibrutinib and anti-CD20 antibody has proven to be clinically highly effective especially for unfit patients with CLL (26, 27) .
As the antibody therapy relies on several independent effector mechanisms antibody-dependent cellular phagocytosis has been previously underestimated (28) .
The effector function of macrophages is highly dependent on interaction within the tumor microenvironment. Therefore, we employed a reliable humanized mouse model of "Double Hit-Lymphoma" for functional elucidation of ibrutinib in antibody combination therapy (17) (18) (19) .
Here, we show that ibrutinib synergizes with multiple monoclonal antibodies in vitro and in vivo via increased macrophage mediated ADCP. We screened the kinase activity of malignant B-cells under ibrutinib, tirabrutinib, and acalabrutinib treatment, identifying Janus Kinase 2 (JAK2) as having significantly reduced activity under ibrutinib, whilst second generation BTKis did not similarly inhibit JAK2 activity.
Finally, we show using CRISPR/Cas9 knockouts and a kinase inhibitor library that loss of JAK2 as well as inhibition using tofacitinib leads to increased ADCP in vitro, whilst superior survival of JAK2 knockout was observed in combination with monoclonal antibody therapy in vivo.
For in vivo experiments 8-14 weeks old male NOD.Cg-Prkdc scid II2rg tm1Wjl /SzJ (NSG, Jackson Laboratory, USA) immunodeficient mice were injected i.v. with 1x10 6 hMB cells diluted in 100 µl PBS (19) . Ten days after injection mice were treated i.p. on three consecutive days with ibrutinib (30 mg/kg), alemtuzumab (day 1 1 mg/kg, day 2 and day 3 5 mg/kg), tirabrutinib (30 mg/kg), or PBS as control. Disease progression was monitored by weekly blood sampling and daily scoring of the mice. Spleen and bone marrow were harvested and dissociated by cell strainers with PBS and lysed with 5 ml ACK lysis buffer for 3 min at RT. For flow cytometry samples were stained with F4-80 APC antibody (BioLegend, San Diego, USA).
The study was approved by the ethical commission of the medical faculty of the University of Cologne (reference no. 13-091) and an informed written consent was obtained from all patients. Primary CLL patient samples were isolated from peripheral blood as previously described (29) . To isolate peritoneal macrophages, wild type and BTK -/-C57BL/6 mice were injected i.p. with thioglycolate and macrophages obtained via peritoneal lavage after four days (18) . BTK -/-C57BL/6 mice were generated by BTK -/mice backcrossed to C57BL/6J background (30) . 
J774A.1 macrophages were cultivated in 96-well plates at 1x10 4 cells per well. After 4 h of incubation at 37°C, 1.5x10 5 hMB "double-hit" lymphoma cells/well were cocultured with respective macrophages. Subsequently, this co-culture was treated for 17 h with tyrosine kinase inhibitors and monoclonal antibodies in combination or as mono treatment. Each condition was performed with five replicates. For determination of ADCP GFP + target cells were analyzed using a MACSQuant flow cytometer. The percentage of ADCP was calculated as follows:
100 -(100 x (cells/µL treated / cells/µL untreated)).
For the generation of conditioned media 1. 
To determine the amount of phagocyted hMB cells by macrophages, J774A.1 were plated out in a density of 1x10 5 cells in 1ml media 4 h prior to addition of target cells. For data analysis Bio Navigator software (PamGene) was used to calculate the cycle and time dependent signals into a single value for each peptide on the chip (exposure time scaling). Furthermore, outliers due to saturation or insufficient antibody binding were excluded. For analysis, data were log transformed calculating the fold change between treated and untreated, and the p-value was calculated using an unpaired Students' two-tailed t-test. A p-value of p≤0.05 was accepted as 8 statistically significant. Individual peptides were matched to their representative kinases using a proprietary database (unpublished; PamGene International B. V., Netherlands). In brief, this database ranked the likelihood of peptides belonging to kinases using public databases such as; PhosphoNet, Reactome and UniPROT.
Volcano plots were produced using the EnhancedVolcano package in RStudio (R version 3.3.1).
All data was evaluated and graphs generated using GraphPad Prism 8. Unless otherwise stated, bar graphs represent the mean ± SD of three biological replicates. 
In order to elucidate the potential synergistic interaction between BTKis and monoclonal antibodies on macrophage-mediated ADCP, we utilized multiple monoclonal antibodies (rituximab, obinutuzumab, alemtuzumab), as well as different types of macrophage effector cells in an ADCP co-culture system ex vivo with the hMB humanized mouse model of "Double-hit" lymphoma as target cells (17, 31) ( Figure 1A ).
Co-treatment of alemtuzumab with serially diluted concentrations of ibrutinib lead to significantly increased antibody-mediated lymphoma cell depletion in a concentration dependent manner ( Figure 1B ). We did not attribute this significant increase to cell toxicity since at these concentrations the cells retained high cell viability (Supp. Figure S1A and B). To verify that ibrutinib specifically enhances phagocytosis of antibody-targeted malignant B-cell lymphoma cells we assessed engulfment of GFP + hMB Double-Hit lymphoma cells into F4/80 + J774A.1 macrophages and detected a rising number of F4/80 + /GFP + cells with increasing ibrutinib concentrations ( Figure   1C ). Furthermore, this effect was independent of the number of macrophages present under ibrutinib treatment (Supp. Figure S1C ). We observed similar significant increases in ADCP performing an independent ex vivo experimental setup using primary murine peritoneal macrophages ( Figure 1D ), whilst also observing concentration dependent antibody-mediated lymphoma cell depletion with the type 1 and 2 anti-CD20 monoclonal antibodies rituximab ( Figure 1E ) and obinutuzumab ( Figure 1F ). Thereby, ibrutinib treatment of hMB cells in vitro induced a moderate but non-significant increase of CD20 and CD52 expression ( Figure 1G ). We furthermore evaluated the effects of ibrutinib on primary leukemic cells from chronic lymphocytic leukemia (CLL) patients. Primary CLL cells pretreated with ibrutinib for 24 h exhibited an increased susceptibility towards alemtuzumab mediated ADCP comparing the ibrutinib treatment with antibody to ibrutinib treatment without antibody therapy ( Figure 1H ). Importantly, here we observe an increase of ADCP using a low ibrutinib concentrations of 0.01 µM. This corresponds to concentrations achieved with oral formulation of 420 mg ibrutinib daily (32) . 
Previously we have shown that chemotherapy in combination with monoclonal antibody treatment induces an ASAP1 leading to increased macrophage-mediated lymphoma cell depletion in vivo (31) . Along these lines, we hypothesized that ibrutinib could be eliciting a similar effect. To analyze the impact of soluble factors released by malignant B-cells, conditioned media from ibrutinib pretreated hMB cells was generated and applied to ADCP co-culture of treatment naïve effector and target cells. Here, we observed a significant increase of ADCP induced by conditioned media obtained from ibrutinib pretreated hMB cells (Figure 2A ). To validate whether ibrutinib was eliciting its action via the effector or target cells, we conducted pretreatment in both J774A.1 and hMB cells, followed by co-culture with treatment naïve cells in the ADCP assay. When we pretreated macrophages with ibrutinib and subsequently added treatment naïve hMB target cells to the ADCP assay we did not detect any significant change in phagocytosis ( Figure 2B ). In contrast, when we applied ibrutinib pretreated hMB cells to treatment naïve macrophages in co-culture, we observed a significant increase in ADCP ( Figure 2C ). This suggests that ibrutinib is eliciting its synergistic interaction with monoclonal antibody mainly in the malignant B-cells.
In comparison to second generation BTKis, ibrutinib is not as highly selective for BTK binding, having affinity for other Tec kinases, such as EGFR and JAK2 (11) .
Therefore, we interrogated whether ibrutinib mediated its effect through covalently binding its main target BTK, leveraging primary peritoneal macrophages from BTK -/mice ( Figure 2D ) and knocking down BTK in hMB cells ( Figure 2E , Supp. Figure   S2A ). In these experiments we observed significant increases in ADCP for BTK -/peritoneal macrophages and no significant difference between wt hMB cells and hMB cells with a knock down in BTK treated with alemtuzumab, suggesting that one of ibrutinib's off-target kinases was responsible for the observed effect. To further confirm that BTK was not responsible for this effect, we conducted ADCP assays in vitro using the second generation BTKis tirabrutinib ( Figure 2F ) and acalabrutinib (Supp. Figure S2B) both showing similar levels of phagocytosis and no toxicity (Supp. Figure S2C and D) . Moreover, combination therapy with alemtuzumab and tirabrutinib in vivo ( Figure 2G ) did not increase overall survival and did not reduce hMB cells or macrophages in spleen and bone marrow. (Supp. Figure S2E-H) . In conclusion, we have identified that the ADCP-enhancing effects of ibrutinib were mediated by targeting the malignant B-cell compartment, which induces a secretory component in the malignant B-cells leading to macrophage activation. Furthermore, we have shown that the observed effect is independent of BTK, therefore we hypothesized that the increased ADCP induced by ibrutinib to be associated with its off-target kinases.
To identify the off-target effects of ibrutinib that might be responsible for the synergistic interaction observed in Figures 1 and 2 Figure 3A and B). In conclusion, this data suggests that JAK2 and JAK3 could be the off-target kinases of 13 ibrutinib, and therefore potentially the responsible kinases for the increased macrophage-mediated phagocytic capacity.
To translate our kinase activity findings back to macrophage mediated ADCP, we generated a modestly sized Tec kinase inhibitor library, conducting concentration dependent ADCP assays in vitro. As expected, inhibition of EGFR (erlotinib; Figure   4A ), SYK (entospletinib; Figure 4B ), and BMX (CHMFL-BMX-078; Supp. Figure   S4A ), lead to no significant increases in phagocytosis. Importantly, inhibition of JAK1/2 via ruxolitinib ( Figure 4C ), JAK2/3 via tofacitinib ( Figure 4D ) and pan-JAK inhibition via SP600125 (Supp. Figure S4B ) all induced significant increases in macrophage mediated ADCP in a concentration dependent manner. Notably, all inhibitors did not display direct cytotoxicity or apoptosis induction to lymphoma target cells in the concentrations used (Supp. Figure S4C -H).
To specifically address the functional implications of JAK2 signaling disruption on lymphoma cell susceptibility to ADCP we generated JAK2 deficient lymphoma target cells (Supp. Figure S4I ). In our ADCP assay JAK2 -/cells showed significantly increased phagocytosis under monoclonal antibody treatment in comparison to JAK2 wildtype cells, which could not be improved by co-treatment with either ibrutinib or tofacitinib ( Figure 4E ). To validate our findings in vivo, we leveraged once again hMB (18, 28) . In this light, previous reports examining ibrutinib in combination with rituximab that did not reveal synergy were mostly focused on NK-cell ADCC or relied on monocyte derived macrophages (20) (21) (22) (23) 35) . Exploring effector cell mechanisms is technically challenging since co-culture systems demand high levels of standardization. However, regarding NK-cell dependent ADCC only modest effects of ibrutinib have been observed (20) or even debated to be inhibitory (21) .
Here we employed two independent macrophage effector cell in vitro models including primary peritoneal macrophages. Moreover, our in vitro ADCP findings were underlined by significantly improved survival in vivo by combination treatment of a humanized mouse model of "Double-Hit" lymphoma (19) . As the used NSG mice 15 are immunodeficient and do not display mature T-cells, B-cells or NK cells, the main effector cells are macrophages which mediate the antitumor effect of alemtuzumab and ibrutinib (36) .
Off-target effects of ibrutinib have been identified recently by independent methodology (34) , likewise using Kinobeads the inhibition of TEC kinase family members and particularly BLK by ibrutinib could be shown (37). However, it remains to be clarified which potential additional kinases beyond BTK are relevant for mediating anti-leukemic effects.
As for the interpretation of clinical trial data it remains to be clarified if ibrutinib combinations with anti-CD20 antibodies rituximab or obinutuzumab are superior to monotherapy in B-cell lymphoma (38) . Nevertheless ibrutinib/ rituximab combination therapy has superior outcome to FC-R as the previous first-line standard therapy of CLL (12) .
Using second generation BTKis we did not observe a synergistic effect, although the combination of acalabrutinib and obinutuzumab was shown to improve progressionfree survival for patients with treatment-naive symptomatic CLL (39) . Here, the high single-agent activity of each drug or ADCP-independent mechanisms of synergy are probably reflecting the superior clinical response in CLL. In our work, we primarily address aggressive Double-Hit lymphoma cells with a potentially less prominent dependence of the malignant B-cell towards sustained BTK signaling.
As ibrutinib has shown clinical activity in MCL and non-GCB DLBCL, ibrutinib in combination with R-CHOP regimens for treatment of first-line DLBCL revealed a clinical benefit only in younger patients (40) . In Waldenström´s Macroglobulinemia (WM) the combination of rituximab and ibrutinib displayed superior progression-free survival and has been FDA-approved (15) . In WM a constitutively activated JAK-STAT signaling pathway has been linked to secretion and disease progression (41) .
As another B-cell receptor signaling related pathway the impact of JAK2-signaling has also been proposed to be relevant in CLL. Here it could be shown that anti-IgM 16 mediated BCR stimulation induces STAT3 activation signaling in CLL (42) . In a clinical trial inhibition of JAK2 by ruxolitinib decreases symptoms (43) 