Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances …following the manufacturer's protocol. In addition, TAM acti-vation was analyzed in E0771 and BMDMs cells. Brieﬂy, 1.0 106

Tumor Biology and Immunology

Pan-TAM Tyrosine Kinase Inhibitor BMS-777607Enhances Anti–PD-1 mAb Efficacy in a MurineModel of Triple-Negative Breast CancerCanan Kasikara1, Viralkumar Davra1, David Calianese1, Ke Geng1, Thomas E. Spires2,Michael Quigley2, Michael Wichroski2, Ganapathy Sriram3,4,5, Lucia Suarez-Lopez3,4,5,6,Michael B.Yaffe3,4,5,7, Sergei V. Kotenko1, Mariana S. De Lorenzo8, and Raymond B. Birge1

Abstract

Tyro3, Axl, and Mertk (TAM) represent a family of homol-ogous tyrosine kinase receptors known for their functionalrole in phosphatidylserine (PS)-dependent clearance of apo-ptotic cells and also for their immune modulatory functionsin the resolution of inflammation. Previous studies in ourlaboratory have shown that Gas6/PS-mediated activation ofTAM receptors on tumor cells leads to subsequent upregula-tion of PD-L1, defining a putative PS!TAM receptor!PD-L1inhibitory signaling axis in the cancer microenvironmentthat may promote tolerance. In this study, we tested combi-nations of TAM inhibitors and PD-1 mAbs in a syngeneicorthotopic E0771 murine triple-negative breast cancer mod-el, whereby tumor-bearing mice were treated with pan-TAMkinase inhibitor (BMS-777607) or anti–PD-1 alone or incombination. Tyro3, Axl, and Mertk were differentiallyexpressed on multiple cell subtypes in the tumor microen-vironment. Although monotherapeutic administration ofeither pan-TAM kinase inhibitor (BMS-777607) or anti–PD-1 mAb therapy showed partial antitumor activity,

combined treatment of BMS-777607 with anti–PD-1 signif-icantly decreased tumor growth and incidence of lung metas-tasis. Moreover, combined treatment with BMS-777607 andanti–PD-1 showed increased infiltration of immune stimu-latory T cells versus either monotherapy treatment alone.RNA NanoString profiling showed enhanced infiltration ofantitumor effector T cells and a skewed immunogenicimmune profile. Proinflammatory cytokines increased withcombinational treatment. Together, these studies indicatethat pan-TAM inhibitor BMS-777607 cooperates withanti–PD-1 in a syngeneic mouse model for triple-negativebreast cancer and highlights the clinical potential for thiscombined therapy.

Significance: These findings show that pan-inhibition ofTAM receptors in combination with anti–PD-1 may haveclinical value as cancer therapeutics to promote an inflamma-tory tumor microenvironment and improve host antitumorimmunity.

IntroductionTyro3, Axl, and Mertk (TAM receptors) comprise a family of

homologous type I receptor tyrosine kinases (RTK) that have beenimplicated as oncogenic kinases overexpressed in human malig-nancies, and more recently as inhibitory or tolerogenic receptorsexpressed on hematopoietic-derived cells [natural killer (NK)cells, dendritic cells, and macrophages] that promote immuno-suppression and resolution of inflammation (1–3). The activa-

tion of TAMs is mediated by homologous endogenous ligands(Gas6 and Protein S; refs. 4–7) that act as hetero-bifunctionalmolecules that bridge TAMswith externalized phosphatidylserine(PS) on apoptotic cells, stressed cells, exosomes, and shed micro-vesicles derived from membrane fragments (8). The activation ofTAMs by Gas6 requires carboxyl-glutamic acid posttranslationalmodification of the Gas6 Gla domain and direct binding to PS foractivity (9, 10). This implies that functionally, TAMs will bemainly active in tissues with constitutively externalized PS, such

1Rutgers University, Biomedical and Health Sciences Center, Department ofMicrobiology, Biochemistry and Molecular Genetics, Cancer Center, Rutgers-New Jersey Medical School, Newark, New Jersey. 2Bristol-Myers Squibb, Prin-ceton, New Jersey. 3Department of Biological Engineering, MassachusettsInstitute of Technology, Cambridge, Massachusetts. 4Department of Biology,Massachusetts Institute of Technology, Cambridge, Massachusetts. 5Center forPrecision Cancer Medicine, David H. Koch Institute for Integrative CancerResearch, Massachusetts Institute of Technology, Cambridge, Massachusetts.6Cancer Research Institute and Department of Medicine, Beth Israel DeaconessMedical Center, Harvard Medical School, Boston, Massachusetts. 7Divisions ofAcute Care Surgery, Trauma, and Surgical Critical Care and Surgical Oncology,Department of Surgery, Beth Israel Deaconess Medical Center, Harvard MedicalSchool, Boston, Massachusetts. 8Rutgers University, Biomedical and HealthSciences Center, Department of Cell Biology and Molecular Medicine, Rutgers-New Jersey Medical School, Newark, New Jersey.

C. Kasikara and V. Davra contributed equally to this article.

Current address for C. Kasikara: Columbia Presbyterian Medical Center, Collegeof Physicians & Surgeons of Columbia University, 630 West 168th Street, PH9-405, New York, NY 10032.

Corresponding Authors: Raymond B. Birge, Rutgers University, Biomed-ical and Health Sciences Center, 205 S. Orange Ave., Cancer Center,Rutgers University, Newark, NJ 07101. Phone: 973-972-4497; Fax: 973-972-5594; E-mail: [email protected]; and Mariana S. De Lorenzo,Phone: 973-972-0822; Fax: 973-972-7489; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-2614

�2019 American Association for Cancer Research.

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as viral infected tissues and in the tumormicroenvironment (11).Indeed, the stromal microenvironment of many solid tumorsdisplay constitutively elevated externalized PS due the combinedhigh apoptotic index of proliferating tumors (12), the occurrenceof metabolically stressed tumor cells and vascular endothelialcells (13, 14), and the release of tumor-derived exosomes fromtransformed cells (15). We have hypothesized that the PS–TAMreceptor axis is constitutively activated in the cancer microenvi-ronment and represents an important target axis in immuno-oncology (9, 11).

The function of TAM receptors as inhibitory receptors thatpromote immune tolerance and resolution of inflammation issupported from both systemic genetic knockout studies inmouse models, by conditional knockout studies in tumor mod-els (16–19), and most recently by pharmacologic inhibitorstudies with small-molecule tyrosine kinase inhibitors to TAMs(20–22). In the former case, TAM knockout mice (either singleKO of Mertk or triple KO of all three TAMs) develop age-dependent autoimmune disease due to the failure to clear apo-ptotic cells under homeostatic conditions (23, 24). However, intumor models, conditional knockout of Mertk on bone marrow–derived monocytes improved tumor immunity in a syngeneicbreast cancer model that correlates with increased inflammatorycytokines and tumor-infiltrating lymphocytes (TIL; ref. 25). Usinga similar genetic strategy, additional studies show that Mertk ontumor macrophages acts as a therapeutic target to prevent tumorrecurrence following radiotherapy, whereby loss of Mertk issufficient to prevent recurrence after C57/Bl6 Mertk (-/-) micechallenged 20 Gy � 1 of focal radiation to the tumor (26).Preclinical studies with pharmacologic agents also showed thatsmall-molecule TAM tyrosine kinase inhibitors, includingBGB324 (27, 28), RXDX106 (29), UNC-2025 (30, 31), andSitravatinib (32) have antitumor activity. Collectively, the impli-cations are that TAMs may act akin to checkpoint inhibitors, asso-called "myeloid checkpoint inhibitors", to alter the cancermicroenvironment, break tolerance, and improve host antitumorimmunity.

In addition to the aforementioned suppressive functions ofTAMs on myeloid expressing cells (NKs, DCs, Macrophages)that assist tumors to evade host antitumor immunity, werecently demonstrated that TAMs, when overexpressed onhuman breast cancer cells promote TAM-mediated epithelialefferocytosis (33) and, in doing so, activate a signaling cascadeto upregulate PD-L1 (33, 34), an inhibitory checkpoint thatbinds to its receptor PD-1 (programmed death receptor-1) on Teffector cells to induce T-cell anergy and tolerance (35). Morerecently, in an AML model, Mertk inhibition by either geneticmanipulation or by Mrx-2843 tyrosine kinase inhibitor signif-icantly decreased PD-L1 and PD-L2 in the tumor microenvi-ronment (36). The inhibitory PD-L1/PD-1 checkpoint hasgained much traction in recent years and motivated the currentdevelopment of anti–PD-1/PD-L1 therapeutic strategies thathave been clinically successful in a variety of indications,including melanoma, NSCLC, and more recently anti–PD-1has shown some effect for breast cancer treatment (37, 38).

On the basis of our previous reports that TAMs, acting as PSsensing receptors, could induce epithelial efferocytosis and thesubsequent upregulation of PD-L1, we propose that in vivo,combinations of PS targeting, TAM therapeutics, and anti–PD-L1/PD-1 may have additive or synergistic activities as combinedtherapeutics. Indeed, prior studies by Gray and colleagues

have shown that combined PS targeting antibodies (upstreamof TAM receptors) with anti–PD-1 function synergistically in asyngeneic breast cancer model (39, 40). The current studyshows that Tyro3, Axl, and Mertk are differentially expressedon several cell subtypes that contribute to the tumor microen-vironment, whereby Axl is preferentially expressed on E0771tumor cells, while macrophages, including peritoneal macro-phages, bone marrow–derived macrophages, and tumor-associated macrophages have higher Mertk/Axl ratios. Subse-quently, we used a combination strategy with a pan-TAMinhibitor, BMS-777607 (41–43), and anti–PD-1 mAb to testthe therapeutic potential in a preclinical model of triple-negative breast cancer. Our data demonstrated that combiningof TAM kinase inhibitor and anti–PD-1 antibody significantlyinhibited tumor growth compared with either single therapyregimen alone or control (vehicle drug) and decreased inci-dence of lung metastasis. Flow cytometry analysis of tumorsrevealed that combination treatment increased infiltrating Tcells and dendritic cells; in contrast to myeloid derived sup-pressor cells (MDSC) whereby infiltration of MDSCs was less inthe tumor microenvironment of combination therapy com-pared with vehicle control. Finally, immune-profiling analysisbased on tumors RNAs demonstrated that combination ofTAM kinase inhibitor (BMS-777607) and anti–PD-1 synergis-tically enhanced expression of proinflammatory cytokines andproimmune cells over control, and addition of BMS-777607to anti–PD-1 treatment downregulated immunosuppressivecytokines expression in tumor microenvironment. Hence, thesestudies support the idea that combination therapies targetingTAMs and PD-1/PD-L1 may have potential to treat humanbreast cancer as immunotherapeutic modalities.

Materials and MethodsCell culture

The murine triple-negative breast cancer cell line E0771(CH3 BioSystems LLC) were maintained in RPMI1640 medium(Sigma-Aldrich) supplemented with 10% v/v heat-inactivatedFBS (Sigma-Aldrich), 100 IU/mL penicillin and 100 mg/mLstreptomycin (Sigma-Aldrich). Cells were grown at 37�C in ahumidified 5% CO2 incubator. After thawing, cells were usedfor up to 5 passages and their authenticities were checked bySTR analysis according to the manufacturer's protocol latest inOctober 2017 (GenePrint 10 System, Promega). Cells areroutinely checked for Mycoplasma contamination.

Measuring TAM surface expression and receptor activationPeritoneal macrophages were harvested from 80 mg/mL

Concavanalin A (Sigma-Aldrich) injected C57BL/6J mice(4 days after Concavanalin injection). Bone marrow–derivedmacrophages (BMDM) were isolated as described below.BMDCs were isolated from C57BL/6J mice by culturing bonemarrow cells in GM-CSF (20 ng/mL) and IL4 (20 ng/mL)for 7 days. Cells were ascertained by FACS to be >70%CD11bþF4/80þ and >70% CD11cþF4/80� for BMDMs andBMDCs, respectively. To determine the surface expression ofTAMs by flow cytometry; E0771 cells, peritoneal macrophages,BMDMs and BMDCs were detached from the plates usingaccutase (Sigma-Aldrich) and then stained with anti-mouseMertk (R&D Systems #AF591), anti-mouse Axl (R&D Systems#FAB8541), and anti-mouse Tyro3 (R&D Systems #MAB759)

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following the manufacturer's protocol. In addition, TAM acti-vation was analyzed in E0771 and BMDMs cells. Briefly, 1.0 �106 E0771 cells or BMDMs were seeded into 35-mm tissueculture plates and then, cells were serum-starved for 6 hours.Later, cells were incubated with Gas6-induced conditionedmedium (�250 nmol/L Gas6) and 5 � 106 apoptotic Jurkatcells (ATCC; prepared as described previously; ref. 33) at 37�Cfor 30 minutes. For inhibition of TAMs activation, 300 nmol/LBMS-777607 (Selleckchem) was added and 293T (ATCC) con-ditioned medium was used as untreated control. After incuba-tion, cells were washed twice with PBS and lysed using HNTGbuffer (20 mmol/L HEPES, 150 mmol/L NaCI, 0.1% TritonX-100, 10% glycerol) as described previously (33). Phosphor-ylated TAM proteins in the detergent lysates were analyzed byimmunoblotting with primary antibodies: phospho-Mertk(Aviva Systems Biology # OASG04503; Fabgennix #PMKT-140AP), phospho-Axl (Cell Signaling Technology #5724),total mouse Mertk (Santa Cruz Biotechnology #sc-365499),total mouse Axl (Santa Cruz Biotechnology #sc-1096), andtotal Tyro3 (Cell Signaling Technology #5585).

IC50 determinationAdvanced Cellular Dynamics tyrosine kinase cell-based assay

(Carna Biosciences) was used for IC50 according to the manu-facturer's protocol; IL3-dependent Ba/F3 cells were transfectedwith human recombinant Tyro3, Axl, Mertk, and VEGFR2 kinasesand treatedwith different concentration of BMS-777607 followedby cell survival and proliferation analysis.

BMDMs and efferocytosis assayBMDMswere generated from tibiae and femurs ofmaleC57BL/

6J mice (The Jackson Laboratory). The bone marrow cells wereflushed with PBS, resuspended in DMEM supplemented with10% heat-inactivated FBS, 20% L929-conditioned medium, andcultured for 7 days. For efferocytosis assay, apoptotic cells werelabeled with pHrodo according to the manufacturer's protocol(Thermo Fisher Scientific). Then, BMDMs were treated withpHrodo-labeled apoptotic cells and Gas6 with/out BMS-777607 for 30 minutes. Cells were washed 5 times with PBS foreliminating nonspecific binding of apoptotic cells and fluorescentintensity of pHrodowas evaluated by using BD FACSCalibur flowcytometry.

qRT-PCR analysis of TAM mRNA in E0771 cells, BMDCs,BMDMs, peritoneal, and tumor-associated macrophages

For tumor-associatedmacrophages, E0771 tumor-bearingmicewere mechanically and enzymatically dissociated and assessed;andmacrophages were gated and sorted according to CD11bþF4/80þ for RNA isolation. Total RNAs from E0771 cells, BMDCs,BMDMs, peritoneal, and tumor-associated macrophages wereisolated using the TRIzol reagent according to the manufacturer'sinstructions. cDNA was obtained using the SuperScript III system(Thermo Fisher Scientific). qRT-PCR for mouse Tyro3, Axl, andMertk was performed using the FAST SYBR Green Master Mix(Thermo Fisher Scientific) with the following primers (IDT):Tyro3: Fwd –GCCTCCAAATTGCCCGTCA, Rev- CCAGCACTGG-TACATGAGATCA.Axl: Fwd–ATGGCCGACATTGCCAGTG,Rev–CGGTAGTAATCCCCGTTGTAGA. Mertk: Fwd – CAGGGCCTT-TACCAGGGAGA, Rev – TGTGTGCTGGATGTGATCTTC. Eachsample was repeated in triplicate and were normalized to theexpression of housekeeping gene, b-actin.

Induced PD-L1 surface expressionTo study IFNg-induced PD-L1 surface expression, E0771 cells

(1 � 106) were seeded in 6-well plates and incubated with100 ng/mL recombinant mouse IFNg (Biolegend) for 48 hours.Untreated and IFNg-treated E0771 cells were harvested andstained with PE-conjugated anti-mouse PD-L1 (BioLegend)according to the manufacturer's protocol and analyzed by flowcytometry.

For Gas6 and apoptotic cell-mediated PD-L1 surface expres-sion, E0771 cells (1 � 106) were seeded in 6-well plates, wereserum-starved for 6 hours and incubated with Gas6-containingconditioned media (�250 nmol/L Gas6) or apoptotic cellsopsonized with Gas6-conditioned medium (�250 nmol/L Gas;ref. 33) with either vehicle (DMSO) or 300 nmol/L BMS-777607 (SelleckChem). 293T-conditioned medium not expres-sing Gas6 was used as untreated control medium. After12 hours, apoptotic cells were washed away with RPMI1640medium twice and E0771 cells were incubated in RPMI1640medium supplemented with 0.5% FBS for an additional12 hours. Subsequently, E0771 cells were collected and stainedwith PE-conjugated anti-mouse PD-L1 (BioLegend) and expres-sion was measured by flow cytometry.

In vivo mouse experimentsE0771 cells (1 � 105) were suspended in 0.15 mL Matrigel

(50 % v/v; Corning) in RPMI1640 medium and injected intothe 9 of 10 mammary fat pads of 7-week-old female C57BL/6mice (n ¼ 8/group) from Charles River Laboratories. Thechimeric anti-mouse PD-1 antibody (4H2; kindly gifted byBristol-Myers Squibb) and the Pan-TAM kinase inhibitorBMS-777607 (SelleckChem) were used as treatment regimens.DMS0 (100 mL/mice/day) and anti-mouse IgG1 isotype anti-body control (5 mg/kg on day 10, 12, 14, and 16) as vehiclecontrol, BMS-777607 (25 mg/kg/day) alone, anti-mouse PD-1(100 mg/mice on day 10, 12, 14, and 16) alone or theircombination were administered via intraperitoneal injectionafter tumors became palpable (on day 10 following tumorimplantation). Doses were selected though preliminary MTDstudies (44, 45). Body weights and tumor growth was assessedevery three days by caliper measurement of tumor diameter inthe longest dimension (L) and at right angles to that axis (W).Tumor volumes were estimated using the formula, L � W �W � p/6. Toxicity and weight loss were not encountered in thestudies. Mice were sacrificed with CO2 on day 28; tumors,lungs, and spleens were collected for further analysis. Whenmetastases were found, the organ was removed and fixed forquantification and histopathology analysis. Metastasis inci-dences were calculated by counting metastatic nodules in thelungs under magnification microscope. Mouse experimentswere performed in accordance with the guidelines and underthe approval from Institutional Animal Care and Use Com-mittee at the Rutgers University, New Jersey Medical.

Flow cytometric analysisHalf of each tumor excised from the mice was physically

dissociated and digested in Hank's balanced salt solution buffersupplemented with 1 mg/mL collagenase (Sigma-Aldrich),0.1 mg/mL hyaluronidase (Sigma-Aldrich), and 200 U/mLDNase type IV (Sigma-Aldrich) for 50 minutes at 37�C andpassed through a 70-mm filter (Falcon). Then, cells were washedwith PBS and treated with red blood cell lysis buffer (Roche)

Targeting TAM Kinases and PD-1 Inhibit Tumor Growth

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for 10 minutes at room temperature. Cells were washedtwice with PBS and stained with antibodies according tothe manufacturer's protocols. Antibodies were used in flowcytometry analysis: PerCP-Cy5.5-conjugated CD45 (BioLegend),APC-conjugated CD4 (eBioscience), FITC-conjugated CD3(eBioscience), PerCP-Cy5.5-conjugated CD25 (BioLegend),PerCP-Cy5.5–conjugated CD11b (eBioscience), PE-conjugatedCD8 (eBioscience), APC-conjugated Ly-6C (BioLegend), FITC-conjugated Ly-6G (eBioscience), and FITC-conjugated CD11c(BioLegend).

NanoString immune-profiling analysisRNAs were isolated from 3 E0771 tumors from each treat-

ment group (vehicle, BMS77607 alone, anti–PD-1 alone, andBMS77607 þ anti–PD-1 combination) by using Direct-zol RNAMiniPrep Plus Kit (Zymo Research) by using manufacturer'sprotocol. All the RNA samples have passed quality control(assessed by OD 260/280) and were subjected to analysis bynCounter murine PanCancer Immune Profiling Panel accordingto manufacturer's protocol at NYU Genomic Center (NanoStringTechnologies). Normalization of raw data was performed usingthe nSolver 3.0 analysis software (NanoString Technologies). Themean of each gene expression (represented in log2) for each groupwas calculated and data were imported to Graphpad Prismsoftware for statistical analysis and graphics. Further advancedimmune-profiling analysis was performed using nSolver 3.0analysis software with nCounter advanced analysis package(NanoString Technologies) with identified immune cell types(classified by Newman and colleagues; ref. 46). Probes used toclassify cell type were as follows: for T cells (Cd2, Cd3d, Cd3e,Cd3g, Cd6, Lck, Cd96, Sh2d1a); for regulatory T cells (Treg)FoxP3; for CD8þ T cells (Prf1, CD8a, Gzmm, CD8b, Flt3lg); fortype 1 T helper cells (Th1; Ctla4, Lta, Ifng, Cd38, Ccl4); for DCs(Cd1e, Cd1b, Ccl17, Ccl22, Cd1a); formacrophage (Cd84, Cybb,Cd163, Cd68); for neutrophils (C1r, Col3a1), and for NK cells(Spn, Xcl2, Ncr1).

Statistical analysisStatistical analysis was performed using GraphPad Prism soft-

ware (GraphPad Software Inc.). In the figures, data represent oneof the triplicate experiments result. Differences between groupswere tested by the two-tailed Student t test or two-way ANOVAfollowed by Tukey post hoc test or Fisher exact test. P values by thet test or Tukey post hoc test or Fisher exact test are shown.Differencebetween groups of P < 0.05 was considered significant.

ResultsTAMs are expressed on E0771 triple-negative breast cancer cellsand on myeloid cells that comprise the tumormicroenvironment

TAMs are expressed on multiple cell types in the tumormicroenvironment where they can function as oncogenic tyro-sine kinases on tumor cells supporting tumor growth, survival,and invasion but also as inhibitory receptors on infiltratingmyeloid-derived cells such as macrophages and DCs, potentiallycontributing as so-called "myeloid checkpoint inhibitors"(1, 47). Here, employing an E0771 orthotopic triple negativebreast cancer mouse model, we observe that TAMs are expressedon both tumor cells as well as myeloid-derived cells thatcontribute to the tumor microenvironment (Fig. 1). When

peritoneal macrophages were elicited by Concanavalin A, orcells from the bone marrow were differentiated with L929conditioned media (containing M-CSF) to become BMDMs;CD11b/F4/80 positive) or with GM-CSF and IL4 to becomeBMDCs (CD11cþ/F4/80 negative), TAMs exhibit differentialexpression in these subsets, as determined by either qPCR(Fig. 1A) or flow cytometry using TAM-specific antibodies todetect surface receptor expression (Fig. 1B). Using a similarapproach to detect TAM expression on E0771 cells, E0771 cellspreferentially express Axl at both the mRNA and protein level,with lesser but detectable Mertk and Tyro3 (Fig. 1C). Finally, inthe tumor-associated macrophages isolated from the E0771-orthotopically proximal transplanted tumors (in this study),Mertk was also preferentially expressed relative to Axl (Fig. 1D),similar to results obtained with BMDMs and peritoneal mac-rophage expression, which showed high Mertk/Axl ratio's inboth subsets (Fig. 1A). These data suggest that TAMs are coex-pressed on both tumor cells, as well as on macrophages and DCsthat comprise the tumor microenvironment.

The aforementioned coexpression of Tyro3, Axl, and Mertk onboth tumor and tumor microenvironment (immune cells)implies that a pan-TAM inhibitor might be expected to directlytarget tumor cells as well as indirectly targeting the tumor micro-environment via the TAM expression on macrophages and DCs.To employ a pan-TAM inhibitor, we tested TAM tyrosine kinaseinhibitors, BMS-777607 and BGB324, using a Ba/F3 tyrosinekinase cell-based IC50 assay, which measures viability of IL3dependent Ba/F3 cells transformed with each tyrosine kinase(Fig. 2A). In this assay, when IL3 is withdrawn from Ba/F3 cells,tyrosine kinase dependent signaling (i.e., TAM activity) overridesIL3 dependency to promote cell survival such that IC50s oftyrosine kinase inhibitors can be measured by a decrease insurvival (shown as percent receptor inhibition; Fig. 2A; ref. 48).Using this assay, we observe BMS-777607 as a broad-based pan-TAM inhibitor with similar IC50s (low to mid nM Kds) towardsTyro3, Axl, and Mertk, while BGB324 showed selectivity towardsAxl. In parallel, employing a CHO-based assay whereby eachintracellular tyrosine kinase domain of TAMs was cloned as anEGFR-TAM chimeric receptor (to normalize postreceptor signal-ing and ligand-dependent kinase activity of each TAM), BMS-777607 also showed efficacy as a pan-TAM inhibitor using phos-phor-TAMs as a readout in cells stimulated with EGF to activateTAM postreceptor signaling (Fig. 2B; ref. 41).

To translate aforementioned in vitro results into a more func-tional outcome, we treated naive peritoneal macrophagesthat express Mertk and Axl (Fig. 1A) with either BMS-777607or BGB324 followed by activation by Gas6 (a pan-TAMligand; Fig. 2C). Notably, 300 nmol/L BMS7706 effectivelyblocked Gas6-inducible pAkt activation (�85%), while Axl-specific tyrosine kinase inhibitor BGB324 only modestly inhib-ited pAkt (�16%; Fig. 2C). These data suggest that BMS-777607 ismore effective than BGB324 to inhibit (pan)-TAM receptor acti-vation onmacrophages. Notably, of other potential BMS-777607targets, including Tyro3 and Axl, and non-TAM tyrosine kinasesFLT, RON, MET, VEGFR2, these receptors are less abundant onperitoneal macrophages relative toMertk (shown are uncorrectedqPCR values; Fig. 2D). However, the role of off-target tyrosinekinases affected by BMS-777607 in vivo (below) cannot be ruledout. Further, to examine effects of BMS-777607 on E0771 tumorcells, 300nmol/L BMS-777607blocked bothGas6-mediated, andGas6-opsonizied AC-mediated activation of Axl on E0771 cells, as

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measured by immunoblotting with a pAxl or pAkt Abs (Fig. 2E).Together, these data suggest that pan-TAM BMS-777607 concom-itantly suppresses TAM activation on both tumor cells andmacrophages.

BMS-777607 blocks macrophage efferocytosis and Gas6-PS–opsonized apoptotic cells; TAM mediated upregulation of PD-L1 on E0771 mouse breast cancer cell lines

To extend TAM kinase activation studies, we pretreatedM-CSF–elicited BMDMsor E0771 cells with BMS-777607 to assayefferocytosis (a macrophage outcome; Fig. 2F) or Gas6/AC-induced PD-L1 upregulation (a tumor cell outcome), respec-tively (Fig. 2G and H). Notably, under these conditions,BMS-777607 suppressedmacrophage efferocytosis in a cell-basedengulfment assay using pHRodo-labeled apoptotic cells (23%inhibition at 1 mmol/L; 77% inhibition at 10 mmol/L; Fig. 2F).Previously, using a series of human breast cancer cell lines, weshowed that TAM-expressing tumor cell lines, when stimulatedwith Gas6-opsonized apoptotic cells, promoted epithelial effer-ocytosis and the upregulation of the checkpoint inhibitor ligandPD-L1 (33). Consistent with these previous findings with humancell lines, when E0771 cells were cocultured with Gas6 (Fig. 2H)

or Gas6-opsonized ACs (5:1 ratio; Fig. 2G, middle panel) for12 hours, followed by washout and incubation for an additional12 hours, surface PD-L1 was upregulated as detected by flowcytometry using a mouse-specific anti–PD-L1 mAb, and thiseffect was potently blocked by BMS-777607 (Fig. 2G, right).Treatment of cells with IFNg was used as a positive control forPD-L1 expression. Hence, in the E0771 cells, BMS-777607 inhib-ited both the early acute tyrosine phosphorylation of TAMs(Fig. 2E) as well as the later upregulation in PD-L1 (Fig. 2G andH). These data suggest that TAMs are activated by Gas6/AC in akinase dependent-manner in E0771 cells, predict a functionalAC!TAM!PD-L1 axis on E0771 cells, and support the rationaleto combine the pan-TAM inhibitors with the checkpointPD-1/PD-L1 inhibitors using in vivo models.

Synergistic antitumor/metastatic effects of pan-TAMBMS-777607 and anti–PD-1 mAbs in the E0771 xenograftmodel

To assess functional relevance in an in vivo model of tumorprogression and immune subversion, E0771 cells were implan-ted into mammary fat pat of C57BL/6 female mice in a longi-tudinal study. Previous studies by Gray and colleagues showed

Figure 1.

Expression of TAMs in E0771 tumor cells and myeloid-derived cells. A, Tyro3, Axl, and Mertk mRNA expression was assessed by qPCR in peritoneal,BMDMs, and BMDCs, as described Materials and Methods. Values represent uncorrected qPCR expression, and data are expressed relative tob-actin mRNA. Data are representative of several independent experiments. B, Flow cytometry analysis (on replicate samples in Fig. 1A) wasperformed to detect Tyro3, Axl, and Mertk surface expression using flow cytometry with TAM-specific, flow-based antibodies. C, qPCR (left) andflow cytometry analysis (right) of surface expressions of TAMs in E0771 cells (tumor cells) used in this study for orthotopic transplantation toC57/BL6 mice. D, Axl and Mertk mRNA expression levels in tumor-associated macrophages (CD11b/F4/80 positive) isolated in the tumor-bearingmice used in this study.






Figure 2.

BMS-777607 is a pan-TAM inhibitor and blocks Axl- and Mertk-dependent signaling in E0771 tumor cells andmacrophages.A, Inhibition of TAMs by BMS77706(left) and BGB324 (right) and assessment of IC50 activities using a Ba/F3 cell-based assay. Following IL3 withdrawal of TAM-expressing Ba/F3 cells, tyrosinekinase inhibitors were titrated to derive IC50s (% receptor inhibition). VEGFRwas used as a non-TAM tyrosine kinase control. B, Schematic illustration of EGFR-TAM chimeric receptors (left). TAM receptors phosphorylation levels were evaluated byWestern blotting after 30minutes EGF treatment with or withoutBMS-777607 (300 nmol/L; more than 10-fold higher than IC50 value) in EGFR-TAM chimeric cell lines (right). (Continued on the following page.)

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antitumor activity in the E0771 model is enhanced by anti–PD-1 therapeutics, implying the tumor microenvironment inthe C57 BL/6 background provides an immune competentmilieu to test checkpoint inhibitors (40). In this study, after10 days, when tumors reached volumes approximately 80 to100 mm3; mice were treated with intraperitoneal injections ofeither vehicle/isotype antibodies alone, with BMS-777607 at aconcentration of 25 mg/kg/day, with anti–PD-1 (5 mg/kg,4 doses every 2 days), or combined BMS-777607 and anti–PD-1 combination as described in Materials and Methods(Fig. 3). Measurements of tumor volume and tumor (wet)weight showed that single regimes of either BMS-77707 oranti–PD-1 mAb partially inhibited the tumor growth comparedwith vehicle or isotype control treatment in the E0771 syngeneicmodel. Notably, however, combinatorial BMS-777607 andanti–PD-1 mAb treatment showed enhanced antitumor effects(volumes and wet weights) compared with monotherapy (P <0.0001; Fig. 3B and C), as well as reduced lung metastaticnodules (Fig. 3D; P < 0.01). Despite efficient suppression oftumor growth, no evidence of weight loss or overt toxicity wasobserved in any treatment group, consistent with previousreports that TAM TKIs (� PD-1) are tolerable in vivo (44, 45).

Combined TAM TKI and anti–PD-1 mAb display increasedtumor-infiltrating lymphocytes

To test whether the aforementioned in vivo antitumor responsesobserved with combinatorial BMS-777607 and anti-PD-1 mAbshowed antitumor immunogenic responses, we dissociated cellsfrom total tumor mass from each treatment group and accessedthe frequency of immune cells subsets, including TILs, by flowcytometric analysis. As indicated in Fig. 4, when we assessedCD45þ in tumor-bearing mice treated with single-agent BMS-777607 or single-agent anti–PD-1 mAb; only the latter showedincreased immune infiltration, suggesting that in the E0771model, pan-TAM TKI inhibitor alone was insufficient to increaseimmune cell infiltration into tumors. However, in combinatori-ally treated mice, the addition of BMS-777607 and anti–PD-1mAb therapy significantly increased CD45þ levels to 56.3 % (P <0.001 to vehicle, P < 0.05 to anti–PD-1 single treatment; Fig. 4A).Furthermore, levels of CD4þ, CD3þ, and CD8þ cells, discretesubpopulations of CD45þ cells, showed a similar trend overvehicle treatment [P < 0.01 (CD3þ), P < 0.001 (CD4þ), P <0.0001 (CD8þ)]. Moreover, combination of BMS-777607 andanti–PD-1 mAb therapy significantly enhanced more CD3þ,CD4þ, and CD8þ subpopulations over single anti–PD-1 therapy,while again, BMS-777607 single treatment did not show signif-icant increase in TILs (Fig. 4B–D). This suggests that general pan-

TAM inhibition, together with anti–PD-1, has a synergistic effectto increase immune infiltration into tumors.

To better understand the molecular mechanisms by whichTAM inhibitors and anti–PD-1 mAb alter the tumor microe-nvironment when used in combination, we profiled RNA expres-sions of genes in specific immune cells based on classificationsdescribed by Newman and colleagues (Fig. 4E–G; ref. 46). Con-sistent with the results of the mechanical dissociation/analysisof cell type frequency in the combination treatment of tumor-bearing mice with BMS-777607 and anti–PD-1 mAb, the expres-sion of genes associated with TILs (Cd2, Cd3d, Cd3e, Cd3g, Cd6,Lck, Cd96, Sh2d1a) was also substantially increased in thetumors compared to anti–PD-1 or BMS-777607 treatment alone(Fig. 4F; P < 0.01 for T cells, anti–PD-1 vs. vehicle; P < 0.001 forT cells, combination vs. vehicle). Similar enhancement effectswere observed in the expression of CD8þ effector T cells (Prf1,CD8a, Gzmm, CD8b, Flt3lg; Fig. 4G) and Treg cells (Fig. 4H;P < 0.01 for CD8þ T-cell combination versus vehicle; P < 0.05 forCD8þ T cells, combination vs. anti–PD-1).

Moreover, when total RNA was isolated from the aforemen-tioned dissected tumors (Fig. 3) and analyzed globally usingNanoString PanCancer Immune Profiling Panel arrays, we alsoobserved the tumors from the combinatorial treatment groups(BMS-777607 þ anti–PD-1) showed an enhanced immunogenicprofile pattern. This profile included enhanced expression ofTNFa (Fig. 5A), IL12 (Fig. 5B), and IFNg (Fig. 5C; P < 0.05 forIL12 and P < 0.01 for IFNg ; combination versus vehicle) andsuppressed expression of immunosuppressive cytokines IL10(Fig. 5D), IL4 (Fig. 5E), and IL13 (Fig. 5F; P < 0.05 for IL10,P < 0.01 for IL4, P < 0.01 for IL13). Interestingly, however, we alsoobserved that PD-L1 expression was increased by combinationtherapy (Fig. 5G), potentially explainedby concomitant increasedIFNg expression seen in combination therapy (Fig. 5C) as it is wellestablished that IFNg can induce PD-L1 expression in cancercells (49). In addition to PD-L1, its receptor PD-1 expression wassignificantly increased by combination therapy (Fig. 5H), whichagainmay be explained by increased total CD45þ cell infiltration.Taken together, combination treatment of BMS-777607 and anti–PD-1 mAb decreased tumor growth by decreasing expression ofsome tumor-promoting (immune suppressor) cytokines andincreasing tumor suppressor (immune activating) cytokines inthe tumor microenvironment.

Although the above-mentioned profiling of TILs and accom-panying cytokines suggested combined BMS-777607 and anti-PD-1 combinatorial treatments induced a T-cell mediated anti-tumor response, we also wanted to expand such profiling toanalyze MDSCs (defined by CD11b, Ly-6C, Ly-6G markers) andDCs (by CD11b, CD11c, and CD8 markers). Compared with

(Continued.) pTAMwas detected using phospho-specific antibodies to each TAM receptor. C, Inhibition of Gas6-induced pAkt (downstream of TAMs) activationin the peritoneal macrophage by BMS-777607 and BGB326. Cells were treated as described in the panel, and after 30minutes, detergent lysates prepared andassayed using pAkt/total Akt. Blots were scanned and densitometry data are shown below the immunoblot. Data are expressed as percent reduction (i.e., pAkt/total Akt ratio). D, Tyro3,Axl, Mertk, FLT3, RON (Mstr1), MET, and KDR (VEGFR2) mRNA expression in peritoneal macrophages was assessed. Values representuncorrected qPCR expression, and data are expressed relative to actin mRNA. E, Effects of BMS-777607 on E0771 cells, where BMS-777607 (300 nmol/L) blocksGas6/Gas-AC induced pAxl and pAkt. F, BMS-777607 blocks efferocytosis in BMDMmacrophages. BMDMswere treated with Gas6-opsonized pHrodo-labeledapoptotic cells with or without BMS-777607 for 30 minutes (1 and 10 mmol/L), after which, efferocytosis levels were evaluated by measuring fluorescenceintensity of pHrodo (percent inhibition is shown). Left, controls (4�C incubation and actin inhibitor cytochalasin D as a negative efferocytosis control).G and H,Effects of BMS-777607 on Gas6 or Gas6-opsonized ACs (5:1 ratio) induced PD-L1 surface expression on the E0771 cells. Flow cytometry analysis of PD-L1 surfaceexpression was performed after 12 hours treatment of Gas6 or Gas6 and apoptotic cells with or without BMS-777607 in E0771 cells. IFNg treatment was used aspositive control (n¼ 3/group).






control treatment,MDSCs'mean value (22.2%), anti–PD-1 aloneand combination treatment showed significant decreased level ofMDSC infiltration to E0771 tumors (14.26%, P < 0.01 for anti–PD-1 treatment; 11.63%, P < 0.001 for combination treatment;Fig. 6A). Similar to CD45þ and subpopulation of CD45þ cellsresult, infiltrated DCs levels (percentage) were significantlyincreased with combination treatment compared with vehicle(P < 0.0001; Fig. 6B). Addition of BMS-777607 to anti–PD-1further demonstrated significant enhancement in DCs levels(RNA expression based; probes: Cd1e, Cd1b, Ccl17, Ccl22, Cd1a)compared with vehicle and anti–PD-1 mAb alone treatment(P < 0.01 combination vs. vehicle; P < 0.05 combination vs.anti–PD-1; Fig. 6C). Taken together, our results in tumor-bearingE0771 mice model showed that anti–PD-1 single treatment was

capable of increasing TILs and combining anti–PD-1 treatmentwith BMS-777607 agents significantly increases infiltrating anti-gen-presenting cells (DCs) further in tumor microenvironment.MDSCs, implicated as immune-suppressors in tumor microenvi-ronment, were decreased in anti–PD-1 and anti–PD-1/BMS-777607 combination treated E0771 mice model. These datasuggest that combination treatment (anti-PD-1 and BMS-777607) enhance antitumor response by promoting immuneactivation in tumor microenvironment.

Effect of TAM kinase and PD-1 inhibition on RNAimmunoprofile of tumor microenvironment

We examined the expression of macrophage (genes analyzed:Cd84, Cybb,Cd163, Cd68; Fig. 6D), neutrophils (genes analyzed:

Figure 3.

Antitumor effect of TAM inhibitor BMS-777607 and anti–PD-1 antibody alone or in combination. A, E0771 tumor-bearing females C57/B16 (n ¼ 8/pergroup) were treated with vehicle, BMS-777607 (25 mg/kg/day) alone, anti-mouse PD-1 (100 mg/mice on day 10, 12, 14, 16) alone (anti–PD-1), or BMS-777607 þ anti–PD-1 combination. Treatments started at day 10 (indicated by arrow) after cell injection, and tumor volumes were measured every 3days. Points, means; bars, SD; �� , P < 0.001 (ANOVA, followed by Tukey range test). B and C, At day 28, before tumors were excised, tumor sizes weremeasured, and tumor average volumes were calculated for each treatment group. Columns, means; bars, SD. C, Wet tumor weights were significantlyreduced in each of the drugs alone and mostly inhibited in the combination-treated-mice. Columns; means; bars, SD. � , P < 0.05; �� , P < 0.01;�� , P < 0.001 and �� , P < 0.0001; P < 0.05 is considered as significant (n ¼ 8/group; Student t test). D, Incidence of lung metastasis was countedfor each group, and differences in the incidence of lung metastasis between treatment groups were compared with Fisher exact test. � , P < 0.05 and�� , P < 0.001 versus vehicle group.

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Figure 4.

Effect of combinatorial therapy with TAM kinase inhibitor and anti–PD-1 antibody on TILs. E0771 tumor-bearing mice were treated with single agents orcombination of BMS-777607 plus anti–PD-1 antibody. At the end of the experiment, three tumors were collected from each treatment group, and single-cellsuspensions were prepared and then stained with specific antibodies against immune cell surface markers. Average percentage for positive surface marker wascalculated by flow cytometry for each group, and data are presented for CD45þ cells (A) and subpopulation of CD45þ cells: CD3þ (B), CD4þ (C), CD8þ (D). RNAswere isolated from the E0771 tumors from treated mice and subjected to further analysis by utilizing NanoString PanCancer Immune Profiling Panel and nSolveradvanced immune-profiling analysis software. Expression profile of CD45 surface markers (E) and profiling tumor-associated immune cell type markers: T cells(Cd2, Cd3d, Cd3e, Cd3g, Cd6, Lck, Cd96, Sh2d1a; F), CD8þ T cells (Prf1, CD8a, Gzmm, CD8b, Flt3lg; G), Treg (FoxP3) cells (H). RNA expression values arepresented in log2 and are graphically represented by GraphPad Prism. Dots, mean (n¼ 3/group); bars, SD. Statistically significant differences between groupswere defined by Student two-tailed t test; � , P < 0.05; ��, P < 0.01; �� , P < 0.001; �� , P < 0.0001 versus vehicle group. n.s., nonsignificant.






Figure 5.

Effect of combinatorial therapy with TAM on cytokine expression. RNAs isolated from E0771 tumors from different treated mice were subjected to NanoStringPanCancer Immune Profiling Panel analysis and nSolver software. Expression profiles of immune activating and immune suppressor cytokines are shown for eachtreatment group (A–F). RNA expression values are presented in log2 and are graphically represented by GraphPad Prism. Dots, mean (n¼ 3/group); bars, SD.Intratumor RNA expression analysis of PD-L1 (G), PD1 (H), and tumor microenvironment cytokines was performed for each treatment group. Statisticallysignificant differences between groups were defined by Student two tailed t-test; � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001 versus vehicle group.n.s., nonsignificant.

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Figure 6.

Effect of combinatorial therapy with TAM on other immune cell types. Average percentage of infiltrating MDSCs surface marker positive (CD11b, Ly-6C, andLy-6G; A) and DCs surface marker positive (CD11c, CD11b, CD8 for flow cytometry analysis; Cd1e, Cd1b, Ccl17, Ccl22, Cd1a for expression analysis; B and C) cells areshown for each treatment group. RNA expressions were analyzed for macrophages (Cd84, Cybb, Cd163, Cd68;D), neutrophils (C1r, Col3a1; E), NK cells (Spn,Xcl2, Ncr1; F), Th1 cells (Ctla4, Lta, Ifng, Cd38, Ccl4; G). RNA expression values are presented in log2 and are graphically represented by GraphPad Prism. Dots,mean (n¼ 3/group); bars, SD. Statistically significant differences between groups were defined by Student two tailed t-test; � , P < 0.05; �� , P < 0.01;�� , P < 0.001; �� , P < 0.0001 versus vehicle group. n.s., nonsignificant.






C1r, Col3a1; Fig. 6E), and NK (genes analyzed: Spn, Xcl2,Ncr1; Fig. 6F) cell markers, combination treatment significantlyaugmented expressions of these three immune cell markers

over vehicle treatment. Similar to increased expression of Tregs(Fig. 4H) in combination treatment (gene analyzed: Foxp3)over vehicle (P < 0.05 for combination vs. vehicle), we observed

Figure 7.

Combination of TAM kinaseinhibitor and anti–PD-1 modulatestumor growth by altering tumormicroenvironment. A,Representative heatmap for celltype abundance in eachtreatment; nCounter advancedanalysis software was used fordrawing heatmap. B, Themodeldemonstrates the mechanism ofaction of TAM kinase inhibitorand anti–PD-1 cancer cells,macrophages, and T cells foundin tumor microenvironment.

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similar enhancements in type 1 T helper (Th1) cells (Fig. 6G)expressions (genes analyzed: Ctla4, Lta, Ifng, Cd38, Ccl4; P < 0.05vs. vehicle group). Representative heatmap for cell type abun-dance was analyzed by nCounter advanced analysis software thatRNA expression for immune cells was used for one sample fromeach group to obtain heatmap analysis (Fig. 7A). Heatmap datademonstrate that combination of treatment induced the abun-dance of infiltrating immune cells compared with vehicle treat-ment (Fig. 7A). According to the tumormicroenvironment Nano-String RNA expression data, these increased immune cells dem-onstrate pro-inflammatory functions by increasing proinflamma-tory cytokine and chemokine expression in dual treatment group.These data suggest that combination treatment of TAMkinase inhibitor and anti–PD-1 mAb enhanced infiltration ofimmune cells into tumor microenvironment compared withvehicle treatment and pan-TAM tyrosine kinase inhibitors arepredicted to have pleotropic effects in the tumor microenviron-ment (Fig. 7B cartoon).

DiscussionPreviously, using TAM-expressing cancer cell lines and a cell

culture system, we showed that Gas6-opsonized apoptotic cells(externalizing PS) activated TAM receptors, induced epithelialefferocytosis (34, 50), and induced up-regulation of the T cellcheckpoint ligand PD-L1 (33). Consequently, these studies pre-dict that PS-positive dying cells and tumors with high apoptoticindexes, in vivo, may have an unanticipated consequence to skewimmune responses that impinge on the PD1/PD-L1 axis. In thepresent study, we extend the previous in vitro studies and showthat combined in vivo administration of anti-PD-1 mAb with apan-tyrosine kinase inhibitor (BMS-777607) enhances tumor-infiltrating lymphocytes and T cell mediated immunity withimproved anti-tumor/anti-metastatic activity compared to singlemono-therapeutics alone. Our study, combined with recentreports by Gray and colleagues showing augmentation in Tcell-mediated immune responses by PS-targeting antibodies plusanti-PD-1 therapy in breast cancer (40), reports by Guo andcolleagues that Axl-specific inhibitor R428 synergizes with PD1therapeutics in colon cancermodels (51), andmost recent reportsbyDu and colleagues that sitravatinib potentiates immune check-point blockage in refractory cancer models (32) support thefurther exploitation of the putative PS!PSR (i.e. TAMreceptors)! PD-L1 axis as an immune checkpoint target in cancerfor further preclinical and future human clinical trials.

Although the present study provides proof-of-concept andsupports the idea that pan-TAM inhibitors, combined withanti-PD-1 or other checkpoint inhibitors, will have therapeuticbenefit as combinatorial regimes in cancer, further mechanisticstudies will be required to identify the repertoire of moleculartargets of BMS-777607 in vivo. For example, in the E0771 triplenegative murine model used in this study, E0771 cells mainlyexpress Axl, although express other TAMs at lower levels. It ispossible that Mertk and Tyro3 can individually impact tumorgrowth and survival, as well as induce PD-L1 up-regulation whenactivated by PS-positive apoptotic cells in other tumors thatexpress these TAMs. Likewise, in recent years, it has been widelyreported that TAMs are differentially and dynamically expressedon a variety of tumor associated myeloid cells, including M2macrophages, DCs, NK cells, and MDSCs, although by common-ality they all act as inhibitory receptors that suppress immune

responses (1). Henceforth, pan-TAM inhibitors likely exert com-plex mechanisms of action on distinct target cell types in thetumor microenvironment, including macrophages, DCs, and NKcells. Adding additional complexity, recent studies also demon-strate that TAMs (as well as Pros1) can be expressed on activatedmemory T cells, and act in a feedback mechanism to limit antigenspecificmemory T cell responses (52, 53). Based on the broad anddynamic expression patterns of TAMs and their ligands on mul-tiple cell types that comprise the tumor microenvironment, infuture studies it will be important to investigate effects of specificTAM antagonists, for example, how Tyro3, Axl, andMertk specificmAbs, or small molecule tyrosine kinase inhibitors, act in differ-ent combinations and in different cancer types.

Despite the board range of potential target cell types for BMS-777607 in the tumor microenvironment, both single therapyBMS-777607 and combined BMS-777607 therapy with anti–PD-1 showed tumor growth inhibition and concomitantenhanced infiltration of tumor-associated lymphocytes, the latterassociated in many cancers, including breast cancer, with betteroverall survival. Indeed, compared with anti–PD-1 treatmentalone, anti–PD-1 and BMS-777607 increased both TILs andintratumoral DCs (tumor antigen presenting cells), and substan-tially shifted the cytokine and chemokine profiles to that typicallyobserved in "hot tumors". This includes increased intratumoralIL12 and IFNg cytokines, while decreased immunosuppressivecytokines (IL10, IL4, IL13, and IL17). Interestingly, we alsoobserved significantly increased intratumoral expression of PDL1,whichmay be counterintuitive given the putative PS!TAM!PD-L1 activation axis that we have proposed. However, it is alsoknown that IFNg has both tumor suppressive and tumor pro-moting activity, the former through the upregulation of MHCclass I (54) and the latter via the upregulation of PD-L1 (55). It ispossible that the increased levels of PD-L1 observed reflect theincreased TILs that produce IFNg , which in turn provide aninductive signal for PD-L1. This might act fortuitously in thismodel, whereby PD-1 mAbs are concomitantly administered.Collectively, in combinationwith anti–PD-1, pan-TAM inhibitorsenhance proinflammatory/antitumor immune cells and cyto-kines in the tumor microenvironment and decrease immunosup-pressive/tumor promoting immune cells and cytokines.

In recent years, the field onco-immunology has gained muchtraction as a therapeutic modality mainly with clinical observa-tions that targeting PD1, and its ligand PD-L1, has producedsignificant clinical benefit in variety of cancers included melano-ma,NSCLC, and renal cell carcinoma.However, it is also clear thatPD-1 blockage is not sufficient to antagonize all resistancemechanisms in the cancer TME. Here, we support proof-of-concept that pan-TAM inhibitors, likely acting as PS receptors,can have synergistic activity with conventional anti–PD-1 thera-peutics and should be further explored in preclinical models andfuture human clinical trials.

Disclosure of Potential Conflicts of InterestM. Quigley has ownership interest (including stock, patents, etc.) in Bristol-

Myers Squibb. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: C. Kasikara, D. Calianese, K. Geng, T.E. Spires,M.B. Yaffe, S.V. Kotenko, M.S. De Lorenzo, R.B. BirgeDevelopment of methodology: C. Kasikara, T.E. Spires






Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Kasikara, V. Davra, K. Geng, M. Wichroski,G. Sriram, L. Suarez-Lopez, M.S. De LorenzoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Kasikara, V. Davra, D. Calianese, K. Geng,T.E. Spires, M. Quigley, M. Wichroski, G. Sriram, M.B. Yaffe, S.V. Kotenko,M.S. De Lorenzo, R.B. BirgeWriting, review, and/or revision of the manuscript: C. Kasikara, V. Davra,D. Calianese, K. Geng, T.E. Spires, M. Quigley, G. Sriram, M.B. Yaffe,S.V. Kotenko, M.S. De Lorenzo, R.B. BirgeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Kasikara, T.E. Spires, R.B. BirgeStudy supervision: T.E. Spires, M.B. Yaffe, S.V. Kotenko, R.B. Birge

AcknowledgmentsWe thank Sukhwinder Singh of Rutgers University Flow cytometry core

facility for flow cytometry technical support and cell sorting. We thank NYU

genomic core facility for NanoString analysis and technical support. Thisresearch was supported by NIH CA 1650771 to R.B. Birge. G. Sriram wassupported by a Mazumdar-Shaw Oncology Fellowship. L. Suarez-Lopez wassupported by a CCFA Research Fellowship #346496. M.B. Yaffe was sup-ported by grants R35-ES 028374 and R01-GM 1044047. S.V. Kotenko wassupported by R01 AI057468 from the National Institute of Allergy andInfectious Diseases.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedAugust 21, 2018; revised January 14, 2019; acceptedMarch 12, 2019;published first March 15, 2019.

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2019;79:2669-2683. Published OnlineFirst March 15, 2019.Cancer Res Canan Kasikara, Viralkumar Davra, David Calianese, et al. CancerPD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast

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Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances …following the manufacturer's protocol. In addition, TAM acti-vation was analyzed in E0771 and BMDMs cells. Brieﬂy, 1.0 106