Tumor Biology and Immunology

Combined VEGF and PD-L1 Blockade DisplaysSynergistic Treatment Effects in anAutochthonous Mouse Model of Small CellLung CancerLydia Meder1,2, Philipp Schuldt1,2, Martin Thelen1,3, Anna Schmitt1, Felix Dietlein4,5,Sebastian Klein6,7, Sven Borchmann1,2,7,8, Kerstin Wennhold1,3, Ignacija Vlasic1,Sebastian Oberbeck1, Richard Riedel1, Alexandra Florin6, Kristina Golfmann1,2,Hans A. Schl€oßer3,9, Margarete Odenthal2,6, Reinhard Buettner2,6,10, Juergen Wolf1,10,Michael Hallek1,10,11, Marco Herling1,2,10,11, Michael von Bergwelt-Baildon1,3,10,H. Christian Reinhardt1,10,11, and Roland T. Ullrich1,2,10

Abstract

Small cell lung cancer (SCLC) represents themost aggressivepulmonary neoplasm and is often diagnosed at late stagewith limited survival, despite combined chemotherapies. Weshow in an autochthonous mouse model of SCLC that com-bined anti-VEGF/anti-PD-L1–targeted therapy synergisticallyimproves treatment outcome compared with anti–PD-L1and anti-VEGF monotherapy. Mice treated with anti–PD-L1alone relapsed after 3weeks andwere associatedwith a tumor-associated PD-1/TIM-3 double-positive exhausted T-cellphenotype. This exhausted T-cell phenotype upon PD-L1blockadewas abrogatedby the additionof anti-VEGF–targetedtreatment. We confirmed a similar TIM-3–positive T-cell phe-

notype in peripheral bloodmononuclear cells of patients withSCLC with adaptive resistance to anti–PD-1 treatment. Mech-anistically, we show that VEGFA enhances coexpression of theinhibitory receptor TIM-3 on T cells, indicating an immuno-suppressive function of VEGF in patients with SCLC duringanti–PD-1-targeted treatment. Our data strongly suggest that acombinationof anti-VEGF and anti–PD-L1 therapies canbe aneffective treatment strategy in patients with SCLC.

Significance: Combining VEGF and PD-L1 blockade couldbe of therapeutic benefit to patients with small cell lungcancer. Cancer Res; 78(15); 4270–81. �2018 AACR.

IntroductionSmall cell lung cancer (SCLC) accounts for 13% to 18% of

primary lung cancer cases and is the most aggressive form ofpulmonary carcinomas, mostly diagnosed at late stages with

systemic metastases. Although current chemotherapies are ini-tially effective in patients with SCLC, responses are typicallytransient and patients succumb to their disease within a fewmonths after diagnosis (1, 2). Therefore, there is a critical needto convert therapy responses into durable remissions and toimprove outcomes in patients with SCLC.

Immune checkpoint blockade using monoclonal antibodiestargeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4),programmed cell-death receptor 1 (PD-1), and programmed cell-death ligand 1 (PD-L1) provided clinical activity in several cancertypes including lung cancer (3, 4). Inhibitory immune checkpointreceptors including lymphocyte-activation gene 3 (LAG-3), T-cellimmunoglobulin and mucin domain 3 (TIM-3), and T-cellimmunoreceptor with Ig and ITIM (TIGIT) block T-cell effectorfunctions and thereby the elimination of tumor cells (5), and theirexpression has been described as a prognostic factor in patientswith SCLC (6). The limitation of immune checkpoint blockade incancer therapy is the activation of different immunosuppressivemechanisms in the tumor microenvironment, which abrogateT-cell effector functions and inhibit the infiltration of tumor-educated T cells into the tumor (7). Because cross-talk between thetumor immune microenvironment and the tumor vasculaturecontributes to tumor immune evasion, combined therapy regi-mens targeting additional immune and/or vascular factors mayprovide sustained and potent antitumor immune responses (7).

As examples of dual immune checkpoint targeting, in advancemelanomas, PD-1 blockade provided an overall response rate

1Department I of Internal Medicine, University Hospital Cologne, Cologne,Germany. 2Center for Molecular Medicine Cologne, University of Cologne,Cologne, Germany. 3Cologne Interventional Immunology, University HospitalCologne, Cologne, Germany. 4Department of Medical Oncology, Dana-FaberCancer Institute, Boston, Massachusetts. 5Cancer Program, Broad Institute ofMIT and Harvard, Cambridge, Massachusetts; US Institute for Pathology, Uni-versity Hospital Cologne, Cologne, Germany. 6Institute for Pathology, UniversityHospital Cologne, Cologne, Germany. 7Else Kr€oner Forschungskolleg ClonalEvolution in Cancer, University Hospital Cologne, Cologne, Germany. 8GermanHodgkin Study Group, Department I of Internal Medicine, University HospitalCologne, Cologne, Germany. 9Department of General, Visceral and CancerSurgery, UniversityHospital Cologne, Cologne, Germany. 10Center for IntegratedOncology Cologne/Bonn, University Hospital Cologne, Cologne; UniversityHospital Bonn, Bonn, Germany. 11Cologne Excellence Cluster in Cellular StressResponses and Aging-associated Disorders (CECAD), Cologne, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Roland T. Ullrich, University of Cologne, Robert-Koch-Street. 21, 50931 Cologne, Germany. Phone: 49-221-478-89771; Fax: 49-221-478-32083; E-mail: roland.ullrich@uk-koeln.de

doi: 10.1158/0008-5472.CAN-17-2176

�2018 American Association for Cancer Research.

CancerResearch

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(ORR) of 33% (8), whereas combined PD-1/CTLA-4 blockaderevealed 72% ORR, but with frequent adverse immune-relatedtoxicities (9). In SCLC, treatment with nivolumab (anti–PD-1)alone provided an ORR of 11% and in combination with ipili-mumab (anti–CTLA-4) an ORR of 25% (CheckMate032). Incontrast to nonsquamous non–small cell lung cancer (NSCLC)PD-L1 expression on tumor cell membranes does not predictresponse to PD-1–targeted therapies in SCLC (10–12). Thus,SCLC still lacks a prediction marker for response to PD-1/PD-L1 blockade wherebymore than 80% of SCLC did not respond tonivolumab (12).

The lack of broad responses to dual immune checkpointblockade in SCLC (12) might refer to additional immunosup-pressivemechanisms in the tumormicroenvironment that are notdirectly triggered by T-cell effector functions. The infiltration ofregulatory T cells (Treg; ref. 13), myeloid-derived suppressor cells(MDSC; ref. 14), and tumor-associated macrophages (TAM) mayreduce the antitumor activity of immune checkpoint blockadesand therefore present a valuable target for novel combinedtherapy approaches (15, 16).

Several studies indicated that anti-VEGF–targeted therapytransforms the tumor immune microenvironment toward animmunosupportive phenotype (17). Dendritic cells (DC) areantigen-presenting cells that take up antigens and present themto T cells. Recent studies showed that VEGF suppresses thematuration of DC precursors and that VEGF blockade improvedDC function and thereby the efficacy of immunotherapy in cancer(18). In addition, high VEGF levels promote the proliferation ofTregs and the expansion of immature myeloid cells, which con-tribute to tumor-associated immunosuppression by suppressingantigen-specific T-cell responses (17). High intratumoral VEGFlevels lead to an abnormal growth of tumor vessels that arecharacterized by hyperpermeable functionally insufficient vessels.This insufficient perfusion is associated with a hypoperfused,hypoxic tumor microenvironment with a high interstitial fluidpressure that impedes effector T-cell infiltration into the tumorand a shift of TAMs toward an immune inhibitory M2-likephenotype with suppressive effector T-cell function (7). Anti-VEGF treatment rescues the expression of adhesion proteins, suchas E-selectin and ICAM-1, on endothelial cells in the tumormicroenvironment and thereby enable effector T-cell migrationinto the tumor tissue (19–22).

Importantly, blocking VEGF/VEGFR signaling was described todirectly regulate the expression of inhibitory immune checkpointreceptors on tumor educated T cells (23). Recent data showed thatreduced VEGFA levels in patients with melanoma are associatedwith response to anti–PD-1-targeted treatment, suggesting thatVEGFA expressionmight be associatedwith response toPD-1/PD-L1 blockade (24).

VEGFA, VEGF receptors (VEGFR), and PD-L1 are highlyexpressed in patients with SCLC (25, 26). Thus, we investigatedthe therapeutic efficacy of combined anti–PD-L1 and anti-VEGF–targeted therapy in a Cre-inducible autochthonous mouse modelof SCLC (27).

Materials and MethodsAnimal experiments

This study was performed in accordance with FELASA recom-mendations. The protocol was approved by the local EthicsCommittee of Animal experiments. The genetically engineered

mouse model of SCLC is driven by a Cre-inducible conditionalRb1 and Tp53 knockout with flox out of exons 2 to 10 in Tp53 andexon 19 in Rb1, as previously described (27). Six-to-8-week-oldmale and female C57BL/6JxFVB/NJx129/Sv mice were anesthe-tized with Ketamin/Xylazin [100 mg/kg/body weight (BW) i.p./0.5 mg/kg/BW i.p.] and 2.5 � 107 pfu Adeno-Cre was appliedintratracheally (28). Viral vectors were provided by the Uni-versity of Iowa Viral Vector Core (http://www.medicine.uiowa.edu/vectorcore). An initial cohort was used to determine sur-vival from the time point of inhalation and estimate a startingpoint for monitoring initial tumor growth (SupplementaryFig. S1). Serial mCT to monitor tumor induction in the therapygroups were started from week 22 after Cre application andtarget lesions were correctly identified from isolated lung tissue(Supplementary Fig. S1). For mCT measurements (LaThetamCT, Hitachi Alcoa Medical Ltd), mice were anesthetized using2.5% isofluran. Histologically, SCLC primary tumors resem-bled human SCLC with regard to cell morphology determinedby hematoxylin and eosin (H&E) stain, proliferation deter-mined by Ki-67 stain and NE marker expression, here CD56.Moreover, SCLC tumors expressed PD-L1 and VEGF (Supple-mentary Fig. S1).

Upon a measurable target lesion, mice were randomly dis-tributed into groups (Supplementary Fig. S1; SupplementaryTable S1) and mice were imaged by mCT once a week. SCLCcohorts comprised five therapy groups, and all therapies weregiven every 3 days simultaneously: (i) vehicle (phosphatebuffered saline; PBS); (ii) IgG (corresponding monotherapyIgGs; Southern Biotech; diluted in PBS); (iii) anti-mouseVEGFA monoclonal antibody (aVEGF, B20-4.1.1-PHAGE,kindly provided by Genentech) (5 mg/kg/BW i.p.); (iv) anti-mouse PD-L1 monoclonal antibody (aPD-L1, clone 6E11,kindly provided by Genentech) (5 mg/kg/BW i.p.); and (v)combined anti-VEGF/anti–PD-L1 (5 mg/kg/BW/5 mg/kg/BWi.p.), where both compounds were applied simultaneously.Reagents were diluted in PBS and obtained from Genentech,which specified the therapy regimen. As a reference group,SCLC-bearing mice were treated with cycles of a standardcombined chemotherapy regimen comprising cisplatin (5mg/kg/BW, 1� per week) and etoposide (10 mg/kg/BW, 3�per week) followed by 2 to 3 weeks of recovery depending ontoxicity and weight loss (Supplementary Fig. S1). Tumorgrowth was monitored by serial mCT whereby the RECISTcriteria v1.1 (29) were adapted to the SCLC model. The min-imal measurable target lesion by mCT scans was adapted to 1mm. Slice thickness was adapted to 0.3 mm. The first dose wasgiven upon target lesion identification and baseline evaluation,maximally 1 day before. Response criteria to evaluate the targetlesion were maintained with regard to diameter fold change.Complete response (CR) referred to a decrease of 100%, partialresponse (PR) was indicated upon a >30% reduction, progres-sive disease (PD) referred to an increase of >20% and/or newlung lesions, and stable disease (SD) was termed upon adiameter change that did not qualify for PR or PD. mCT datawere analyzed using OsiriX-DICOM viewer (aycan Digitalsys-teme GmbH). Progression-free survival (PFS) and overall sur-vival (OS) of the therapy groups and the predictive additiveprobability of survival of both monotherapy-treated cohortswere analyzed as follows: Let pA ðtÞ, pB ðtÞ, pA;B ðtÞ, and pCntrl ðtÞdenote the probability of survival for t 2 ½0;¥Þ under therapywith compound A, B, their combination or vehicle solution,

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respectively. In order to determine whether the combination oftwo drugs A and B had a synergistic impact on survival, wecalculated the expected additive curve as

pAþB tð Þ :¼ min 1; pCntrl tð Þ þ pA tð Þ � pCntrl tð Þj j�0 þ pB tð Þ � pCntrl tð Þj j�0Þ�

where pAþBðtÞdenotes the expectedprobability of survival, assum-ing the combination effect of drugs A and B is additive, andjaj�0 :¼ maxð0; aÞ. Based on this definition, we calculated theexpected number of events at each time point of pAþBðtÞ byinverting the Kaplan–Meier statistics, assuming equal cohort sizesbetween combination and single-agent cohorts. Based on theseevents, we finally compared the expected additive survival ratepAþBðtÞ and the observed survival rate pA;B ðtÞ under drugs A and Bin combination, using a Mantel–Cox test. Scripts are availableupon request.

Flow cytometryOrgans of mice were harvested, and cells were isolated by

mechanical dissociation using 40-mm cell strainers (BD Falcon).Red blood cells were lysed byACK lysis buffer (Life Technologies),and cells were washed with PBS. Purified primary cells and T cellswere stained for 30 minutes at 4�C for flow cytometry usingantibodies against following targets and isotype controls, bothobtained fromBioLegend if not otherwise specified: LAG-3 (FITC,C9B7W, Thermo Scientific), CTLA-4 (PE, UC10-4B9), CXCR3(FITC, CXCR3-173), CCR4 (PE-Cy7, 2G12), FOXP3 (PE, MF-14), IFNg (Alexa Fluor 700, XMG1.2), TIM-3 (PE, RMT3-23;PerCP-Cy5.5, B8.2C12), CD4 (PE-Dazzle594, GK1.5), CD45(PerCP-Cy5.5, Alexa Fluor 700, APC-Cy7, 30-F11), CD3 (PE-Cy7,Alexa Fluor 700, 17A2), PD-1 (APC, 29F.1A12), CD8a (FITC,Pacific Blue, 53-6.7), CD11c (PE-Dazzle594, N418), F4/80 (AlexaFluor 700, BM8), PD-L1 (PE-Cy7, 10F.9G2), PD-L2 (eBioscience,FITC, 122), H-2Kb (Pacific Blue, AF6-88.5), galectin-9 (PE,108A2), CD56 (R&D Systems, APC, 809220), Rat IgG2aK (FITC,PE, PerCP-Cy5.5, APC, Alexa Fluor 700), PE-Dazzle594ArmenianHamster IgG (PE-Dazzle594), Rat IgG2bK (PE-Cy7), and mouseBALB/c IgG2aK (Pacific Blue). In addition, APC-Cy7-conjugatedfixable viability dye (eBioscience) or the Zombie Aqua FixableViability Kit (BioLegend) was used.

Purified human T cells were stained in 50 mL volume for 20minutes at 4�C for flow cytometry using antibodies againstfollowing targets, obtained from BioLegend if not otherwisespecified: CD45 (FITC, HI30), TIM-3 (PE-Dazzle594, F38-2E2),CD3 (PerCP-Cy5.5, SK7), CD4 (PE-Cy7, SK3), PD-1 (APC,EH12.2H7), CD8a (Alexa Fluor 700, SK1), CD69 (APC-Fire750, FN50). In addition, a Pacific Orange–conjugated fixableviability dye (Zombie Aqua) was used. Flow cytometry formurine and human cells was performed on a Gallios 10/3(Beckman Coulter), and data were analyzed using FlowJo(TreeStar v7.6.1).

T-cell stimulationMurine T cells were isolated from harvested spleens of mice

harboring SCLC using MojoSort Mouse CD3 T-cell Isolation(BioLegend) according to the manufacturer's protocol. T cellswere cultured in 96-well flat bottom plates (Sarstedt) and stim-ulation was performed for 24 hours using 5 mg immobilized anti-CD3 (BD Pharmingen), 2 mg/mL soluble anti-CD28 (BD Phar-mingen), 10 ng/mL murine IFNg (PeproTech) and 50 ng/mL

murine VEGF165 (PeproTech), also known as VEGFA. Humanperipheral blood mononuclear cells (PBMC) were isolated frompatients with SCLC blood between 2 and 4 weeks after the lastdose received by density gradient centrifugation using Pancoll(Pan Biotech, density 1.077 g/L). T cells were purified bymagneticcell sorting using MojoSort Human CD3 T-cell isolation (BioLe-gend) according to the manufacturer's protocol for column-freeisolation. T cells were cultured in 96-well flat bottom plates(Sarstedt) under humanized conditions and stimulated for 24and 72 hours using 5 mg/mL immobilized anti-CD3 (BioLegend),2 mg/mL soluble anti-CD28 (BioLegend), and 50 ng/mL humanVEGF165 (PeproTech).

IHCMurine organs were harvested and fixed in 4% PBS-buffered

formalin for paraffin embedding. Three-micrometer tissue sec-tions were deparaffinized and IHC was performed using theLabVision Autostainer-480S (Thermo Scientific) staining withH&E, primary antibodies against KI-67 (Cell Marque, SP6),CD31 (BD Pharmingen, MEC13.3), CD56 (Abcam, polyclonal,ab95153), PD-L1 (proteintech, polyclonal, 17952-1-AP), VEGF(Santa Cruz Biotechnology, A-20), CD4 (Abcam, EPR19514),CD8 (Abcam polyclonal, ab203035), FOXP3 (Novus Biologi-cals, polyclonal, NB100-39002), and the Secondary-Histofine-Simple-Stain (SHSS) antibody detection kit (Medac). Slideswere scanned by the Panoramic-250 slide scanner (3D His-tech). CD31 staining was used to determine microvessel den-sity. Briefly, five representative 20� enlarged fields wereextracted from each slide using the Panoramic Viewer Software.A customized script of ImageJ (National Institutes of Health)was used to identify CD31-positive structures. The number ofstructures with a minimum size of 30 pixels was counted per20� enlarged field and averaged across all fields for each slide.Human SCLC was diagnosed based on histologic examinationby trained lung pathologists. Pictures were acquired with aLeica-DM-5500 B microscope. Primary antibodies against PD-L1 (28-8, Abcam), TIM-3 (D5D5R, Cell Signaling Technology),galectin-9 (D9R4A, Cell Signaling Technology), CD56 (123C3,Zytomed), synaptophysin (SP11, Thermo Fisher Scientific),and chromogranin A (DAK-A3, Dako) were used. Secondaryantibodies were purchased from ImmunoLogic (Bright-Visionþ) and staining was performed using the LabVisionAutostainer 480S (Thermo Scientific).

EthicsAll human subject research was performed in strict accordance

with approved protocols by the local ethics committee of theUniversity Hospital Cologne and with the recognized ethicalguidelines of the Declaration of Helsinki. Blood samples (refer-ence number 17-130) and tumor tissue (reference number 10-242) were obtained during routine clinical procedures frompatients diagnosed based on the World Health Organizationclassification of lung tumors (30) providing written informedconsent for additional tissue collection.

Statistical testsStatistical analyses were done using Prism (GraphPad V5.0)

and SPSS (IBM, V24.0). Error bars indicated standard error of themean (SEM). P values <0.05 were regarded as significant andindicated in the figures: �, P � 0.05; ��, P � 0.01; ���, P � 0.001.

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ResultsCombined anti-VEGF/anti–PD-L1-targeted therapysynergistically improves PFS and OS in SCLC

We performed a therapeutic study in an autochthonous mousemodel of SCLC in which tumors are induced upon Cre-mediatedbiallelic deletion of Rb1 and Tp53. We recorded the clinicopath-ologic parameters of SCLC bearing mice listed according to theapplied therapy regimens (Supplementary Table S1). Mice wererandomized and systemically treated with vehicle, correspondingIgGs, anti-VEGF, anti–PD-L1, and with the combination of anti-VEGF and anti–PD-L1 targeting. Anti-VEGF monotherapy inSCLC-bearing mice did not improve PFS and OS (1 week, 3weeks, respectively) in comparison with vehicle-treated mice(1 week, 3 weeks, respectively) and IgG-treated mice (1.5 weeks,2.5weeks, respectively; Fig. 1AandB). Treatmentwith anti–PD-L1alone significantly improved PFS (2weeks, P¼ 0.0061) andOS (4weeks, P ¼ 0.0008) compared with the vehicle group. Moststrikingly, combined anti-VEGF/anti–PD-L1 inhibition led to asubstantial improvement in median PFS (3 weeks) and OS(6 weeks) in comparison with anti–PD-L1 monotherapy (PFS:P ¼ 0.0166, OS: P ¼ 0.0231; Supplementary Tables S2–S5). Todecipher whether the combination of anti-VEGF/anti-PD-L1–targeted therapy results in synergistic treatment effects, we calcu-lated predicted additive PFS and OS curves as described in theMaterials and Methods. Using Prism Mantel–Cox test (Supple-mentary Tables S2 and S3), we compared the predicted additivecurves ofOS and PFSwith the observed survival curves of the anti-VEGF/anti–PD-L1 combined therapy group (PFS: 0.0119,OS: P¼0.0316; Fig. 1C and D). Because we determined a significantdifference between the predicted additive and the combinationtherapy curves, the therapeutic effect on survival of anti-VEGF andanti–PD-L1-targeted therapy, which were administered simulta-neously,wasdefined as synergistic effect.We further comparedOSdata of combined anti-VEGF/anti–PD-L1 treatmentwith standardcombined cisplatin/etoposide chemotherapy. Of note, OS of themice with SCLC treated with combined anti-VEGF/anti–PD-L1therapywas better than the observedOSupon standard combinedchemotherapy regiments (median OS: 5 weeks, 6 weeks, respec-tively, P ¼ 0.1312; Supplementary Fig. S1).

We next assessed the change in target lesion diameter in alltherapy groups after 1 and 2 weeks of treatment using mCTanalysis. After 1 week of treatment, we found one SD in SCLC-bearing mice treated with anti-VEGF alone (20.0%, 2 of 10),while upon anti–PD-L1 monotherapy this was seen in 78.6%(11 of 14), and in 92.3% (12 of 13) with combined anti-VEGF/anti–PD-L1 treatment. A PR was found in 7.1% of mice (1 of14) treated with anti–PD-L1 and 7.7% of mice (1 of 13) treatedwith combined anti-VEGF/anti–PD-L1. However, already after1 week, 80.0% of mice (8 of 10) treated with anti-VEGFmonotherapy, 50% of mice (3 of 6) treated with IgG controlsand 100% of vehicle-treated mice (12 of 12) showed PD (Fig.1E). After 2 weeks of treatment, SD was detected upon anti–PD-L1 monotherapy in 42.9% (6 of 14) and upon combinedanti-VEGF/anti–PD-L1 in 46.2% (6 of 13). Of note, a PR wasonly determined in SCLC-bearing mice treated with combinedanti-VEGF/anti–PD-L1 (15.4%; 2 of 13). PD was determined inall vehicle-treated (12 of 12), all IgG-treated (6 of 6), and all ofthe anti-VEGF–treated mice (10 of 10). Of anti–PD-L1 mono-therapy-treated mice, 42.9% (6 of 14) showed a PD, while only23.1% (3 of 13) of combined anti-VEGF/anti–PD-L1-treatedmice showed progressive tumors (Fig. 1F). Representative serial

mCT measurements indicated a PR upon combined anti-VEGF/anti–PD-L1 therapy followed by an SD at 4 weeks and a finalPD after 7 weeks of treatment (Fig. 1G).

Taken together, treatment of SCLC-bearing mice with com-bined anti-VEGF and anti–PD-L1-targeted therapy synergisticallyimproves PFS and OS compared with anti–PD-L1 and anti-VEGFalone.

Anti–PD-L1 therapy induces an exhausted T-cell phenotypethat is diminished in mice with combined anti-VEGF andanti–PD-L1 treatment

We next analyzed the impact of anti–PD-L1, anti-VEGF, andcombined anti-VEGF/anti–PD-L1 treatment on tumor-infiltratingT cells. First, the localization of CD4þ, CD8þ, and FOXP3þ T cellswas examined using IHC (Fig. 2A). In vehicle-treated mice withSCLC, T cells did not accumulate in the pulmonary tissue aroundthe tumor and did not infiltrate the tumor tissue. In anti–VEGF-treatedmice, tumorswere infiltrated by fewCD4þT cells, wherebyCD8þ and FOXP3þ T cells remained at the tumormargin. In anti–PD-L1-treatedmice, CD4þ andFOXP3þT cells accumulated in thepulmonary tissue at the tumor margin but did not invade tumortissue. In SCLC-bearing mice treated with combined anti-VEGF/anti–PD-L1 CD4þ T cells and few FOXP3þ and CD8þ T cellsinfiltrated tumor tissue. We also calculated CD31-positive tumormicrovessels in progressed SCLC lesions upon therapy and didnot detect significant differences in microvessel density (Supple-mentary Fig. S2).

Second, we generated single-cell suspensions from primarytumors and immunodetected tumor cells and immune cells,including T cells (CD45þ CD3þ). We found a significantlyincreased ratio of pan-immune cells (CD45þ) to tumor cells(CD45� CD56þ), but the fraction of T cells within the immunecell compartment, the CD4/CD8 ratio, and IFNg expression werenot significantly altered upon anti–PD-L1 and combined anti-VEGF/anti–PD-L1-targeted therapy compared with vehicle (Sup-plementary Figs. S3 and S4). However, we found a significantincrease in the fraction of Tregs (CD4þ FOXP3þ) in tumors thatprogressed upon combined anti-VEGF/anti–PD-L1 therapy (Sup-plementary Fig. S5).We further analyzed the fractions of Thelper1(Th1) and Th2CD4þ T cells using CXCR3 and CCR4markers. Theratio of Th1 (CD4þ FOXP3� CXCR3þ) and Th2 (CD4þ FOXP3�

CCR4þ) cells was not significantly affected using anti-VEGF andanti–PD-L1-targeted therapies (Supplementary Fig. S5).

With regard to CD8þ T cells, their assembly at the site of thetumor was significantly increased upon the initial response tocombined anti-VEGF/anti–PD-L1 treatment (Supplementary Fig.S6), which disappeared upon PD.

To elucidate mechanisms of adaptive resistance, we analyzedimmune checkpoint expression in tumor-infiltrating lympho-cytes. Generally, the T-cell function is mediated by receptor–ligand interactions, which may have stimulatory or inhibitoryeffects. An exhausted T-cell phenotype is indicated by simul-taneous upregulation of at least two inhibitory receptors,such as PD-1 and TIM-3 (3). Herein, we found upregulationof the immune checkpoints TIM-3, LAG-3, and PD-1 on CD4þ

and CD8þ T cells in tumors with adaptive acquired resistanceagainst anti–PD-L1 therapy (PD-1/TIM-3: CD4þ P ¼ 0.0081 andCD8þ P ¼ 0.0071; PD-1/LAG-3: CD4 P ¼ 0.0082 and CD8 P ¼0.0395; Fig. 2B–E). CTLA-4 and PD-1 double-positive T-cellfractions were not increased upon acquired anti–PD-L1 resistance(Supplementary Fig. S7). This exhausted T-cell phenotype was

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significantly increased in tumors that progressed during anti–PD-L1 treatment in comparison with anti–PD-L1-treated mice withPR and SD, which represents a true phenotype acquisition (CD4þ

P ¼ 0.0310 and CD8þ P ¼ 0.0062; Supplementary Fig. S6).Moreover, we found that the exhausted T-cell phenotypewas locally associated with the presence of tumor cells (Supple-mentary Fig. S8).

Interestingly, the PD-1/TIM-3–exhausted T-cell phenotypewas significantly rescued by combining anti–PD-L1-targetedtherapy with anti-VEGF therapy (CD4þ P ¼ 0.0389; CD8þ

P ¼ 0.0411, respectively), whereas the PD-1/LAG-3–exhaustedphenotype was not rescued (Fig. 2B–E). We also showed thatthe exhausted T-cell phenotype was not dependent on IFNg(Supplementary Fig. S9). Moreover, VEGF knockout in SCLCtumors did not enhance response to aPD-L1 treatment (Sup-plementary Fig. S10).

As TIM-3 was upregulated upon progression during anti–PD-L1-targeted treatment, whichwas again abrogated by the additionof anti-VEGF therapy, we hypothesized that VEGF/VEGFR signal-ing induce TIM-3 expression on tumor-associated T cells. Wefound that VEGFR1 was upregulated on tumor-associated CD8þ

T cells (Supplementary Fig. S11). Thus, these data might indicatethat TIM-3 expression is regulated by VEGF-VEGFR1 in CD8 Tcells. However, the VEGF-induced signaling pathway that regu-lates TIM-3 expression in CD-8 cells remains elusive.

Taken together, combining anti-VEGF to anti–PD-L1-targetedtherapy rescued T-cell exhaustion, which was observed as anacquired resistance mechanism to PD-L1 blockade in SCLC.

Increased galectin-9 expression in TAMs upon PD-L1 treatmentWe further investigated the expression of PD-L1 (31), PD-L2

(32), and galectin-9 (33) in the tumor microenvironment. Theyrepresent prominent ligands for PD-1 and TIM-3, respectively,and trigger immunotolerance and tumor immune evasionthrough the abrogation of IFNg signaling and the induction ofeffector T-cell apoptosis (31–33).

We analyzed the expression of these ligands on tumor-associated DCs (TADC; CD45þ CD56� F4/80� CD11cþ),tumor cells (CD45� CD56þ), and TAMs (CD45þ CD56� F4/80þ). The expression of galectin-9 was significantly increasedon TAMs in SCLCs that progressed during anti–PD-L1 andcombined anti-VEGF/anti–PD-L1 therapy (measured by meanfluorescence intensity compared with vehicle, anti–PD-L1: P ¼0.0069, anti–PD-L1/anti-VEGF: P ¼ 0.0212, respectively), butwas not significantly altered on TADCs and tumor cells (Fig. 3Aand B; Supplementary Fig. S12). In human SCLC patientsamples without anti-PD-1/anti–PD-L1 treatment, galectin-9was detected on tumor cells (3 of 18), TAMs and lymphocytes(8 of 18) and frequently coexpressed with TIM-3 (7 of 8) butnot with PD-L1 (Supplementary Fig. S13; Supplementary

Tables S6 and S7). PD-L1, which was shown to be significantlyexpressed on tumor cells and within the tumor microenviron-ment of SCLC (26), was significantly reduced on TADCs (anti–PD-L1: P > 0.001, anti–PD-L1/anti-VEGF: P ¼ 0.002, respec-tively), tumor cells (anti–PD-L1: P ¼ 0.0111, anti–PD-L1/anti-VEGF: P ¼ 0.0220, respectively), and TAMs (anti–PD-L1: P ¼0.0002, anti–PD-L1/anti-VEGF: P¼ 0.0009, respectively), uponanti–PD-L1 treatment in the monotherapy and combined anti-VEGF/anti–PD-L1 therapy cohort (Supplementary Fig. S12).PD-L2 was not expressed on TADCs and TAMs, but at lowlevels on tumor cells. However, PD-L2 was not differentiallyexpressed in SCLC following the applied therapy regimens(Supplementary Fig. S12). Furthermore, we analyzed theexpression of MHC class I on TADCs, TAMs, and tumor cells,as genomic aberrations in B2M resulting in MHC class I losswere identified as potential resistance mechanism to PD-1blockade in melanoma (34). However, MHC class I was notdifferentially expressed between any of the cell types in ourSCLC therapy cohorts (Supplementary Fig. S12). We also ana-lyzed the ratio of TAMs and TADCs within the immune cells butdid not identify significant alterations upon application of thedifferent therapy regimens (Supplementary Fig. S3).

Because immune checkpoint receptors were recently discov-ered on TAMs, abrogating their antitumor function and directlycontributing to the response to immune checkpoint blockade(35, 36), we investigated the expression of PD-1, TIM-3, LAG-3,and CTLA-4 on TAMs in mice with SCLC (Supplementary Fig.S14). We found that immune checkpoint receptor expressionwas induced on TAMs upon acquired resistance to PD-L1blockade (PD-1 P < 0.0001; TIM-3 P ¼ 0.1053; LAG-3 P ¼0.0064; CTLA-4 P ¼ 0.0015). Combining anti–PD-L1/anti–VEGF-targeted therapy reduced LAG-3 and CTLA-4 expressionsignificantly (P ¼ 0.0012; P ¼ 0.0003, respectively) comparedwith anti–PD-L1 monotherapy. Taken together, TAMs mightpresent an exhausted phenotype like T cells upon acquiredresistance to PD-L1 blockade and may contribute to the pro-longed survival of mice with SCLC achieved by combined anti-VEGF/anti–PD-L1 treatment.

VEGF significantly upregulates TIM-3 on CD8þ T cellsisolated from human PBMCs upon acquired resistance tonivolumab

To validate our preclinical findings in human patient samples,we isolated PBMCs from patients with SCLC that progressedduring nivolumab (anti–PD-1) treatment. We investigatedPBMCs of two cohorts of patients with SCLC: those treated withirradiation and chemotherapy alone (RC) or those treated withRC followed by the immune checkpoint inhibitor nivolumab(RCI). Confirming our preclinical data derived from mice withautochthonous SCLC, TIM-3 was significantly upregulated on

Figure 1.Combined anti-VEGF/anti–PD-L1-targeted therapy synergistically improves PFS and OS in SCLC. SCLC-bearing mice were treated with vehicle (black; n ¼ 12), IgGcontrol (violet; n ¼ 6), anti-VEGF monotherapy (aVEGF, orange; n ¼ 10), anti–PD-L1 monotherapy (aPD-L1, green; n ¼ 14), and combined anti-VEGF/anti–PD-L1therapy (combi, red; n ¼ 13) and serially imaged by mCT. A and B, PFS and OS were determined from the five therapy groups. Statistical analysis was doneusing the Prism Mantel–Cox test (ns, not significant; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001; black star, compared with vehicle; violet star, compared with IgG; orangestar, compared with aVEGF; green star, compared with aPD-L1; blue star, compared with additive). The corresponding P values, the c2 value, and the hazardratios are listed in Supplementary Tables S2–S5. C and D, The predicted additive PFS and OS survival curves (blue) were calculated as described from the anti-VEGFand the PD-L1 monotherapy group and compared with the PFS and OS curves of the combined anti-VEGF/anti–PD-L1 therapy group of A and B (combi, red)using Prism Mantel–Cox test. P values are indicated. c2 values and hazard ratios are listed in Supplementary Tables S2–S5. E and F, Change in target lesion diametercalculated from all therapy groups after 1 week (E) and after 2 weeks (F) of treatment. Striped columns refer to animals that died before 2 weeks of treatment,so the last determined value was plotted. PD, SD, PR, and CR according to described mouse adapted RECIST v1.1 criteria. G, Serial mCT measurements of onerepresentative mouse per therapy group. Target lesion diameter is marked green. H, heart; D, diaphragm; †, dead.

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CD8þ andCD4þ T cells of peripheral blood of patients with SCLCwho progressed following response to anti–PD-1 therapy. Asexpected, PD-1 on these peripheral blood T cells was down-regulated due to anti–PD-1 treatment (Fig. 4A–C).

Following our postulate that VEGF induces TIM-3 expression intumor-associated T cells, we investigated the effect of VEGFstimulation on T cells derived from PBMCs of the above-men-tioned patient cohorts. In line with our hypothesis, the fraction of

Figure 2.

Anti–PD-L1-resistant SCLC show significantlyincreased PD-1/TIM-3 double-positive CD8þ andCD4þ T cells. SCLC-bearing mice were treated withvehicle (n ¼ 15), anti-VEGF monotherapy (aVEGF;n¼ 10), anti–PD-L1monotherapy (aPD-L1; n¼ 11), andcombined anti-VEGF/anti–PD-L1 therapy (combi;n ¼ 10), and endpoint analysis was performed usingIHC and flow cytometry. A, CD4, CD8, and FOXP3stains on FFPE SCLC tissue by IHC. Images weretaken at �20 magnification. Bars, 100 mm. B, CD45þ

CD3þ CD8þ T cells were analyzed for PD-1 and TIM-3expression. Dot plots of one representativeexperiment per therapy group are shown. C, CD45þ

CD3þ CD4þ T cells were analyzed for PD-1 and TIM-3expression. Dot plots of one representativeexperiment per therapy group are shown. D, CD45þ

CD3þ CD8þ T cells were analyzed for PD-1 and LAG-3expression. Dot plots of one representativeexperiment per therapy group are shown. E, CD45þ

CD3þCD4þ T cells were analyzed for PD-1 and LAG-3expression. Dot plots of one representativeexperiment per therapy group are shown. Statisticalanalysis was done using the Student t test (ns, notsignificant; � , P < 0.05; error bars, SEM).

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Figure 3.

Anti–PD-L1-resistant SCLC show higher galectin-9expression in TAMs. SCLC-bearing mice were treatedwith vehicle (n ¼ 4), anti-VEGF monotherapy(aVEGF; n ¼ 4), anti–PD-L1 monotherapy (aPD-L1;n ¼ 5), and combined anti-VEGF/anti–PD-L1 therapy(combi; n ¼ 4). Upon detection of PD based on mCTmeasurements and mouse-adapted RECIST v1.1criteria, endpoint analysis was performed using flowcytometry. A, Lysates from primary tumor materialwere stained for viable (viaþ) immune cells (CD45þ)and nonimmune cells (CD45�), which were used toidentify CD56þ SCLC cells. T cells (CD3þ) wereidentified within the CD45þ gate. The CD45þ CD3�

cells were used to identify TAMs using anti-F4/80 andanti-MHCI. TheCD45þCD3�F4/80� cellswere used toidentify TADCs by anti-CD11c. B, Relative galectin-9expression of TAMs, determinedbymean fluorescenceintensity (MFI), was normalized to IgG control.Histograms of one representative experiment pertherapy group are shown. Statistical analysiswas doneusing the Student t test (ns, not significant; � , P < 0.05;�� , P < 0.01; error bars, SEM).

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Figure 4.

Stimulation with VEGF significantlyincreases the fraction of PD-1/TIM-3double-positive CD8þ T cells. PBMCsfrom patients with SCLC who receivedradiochemotherapy (RC; n ¼ 3) orradiochemotherapy, followed bytreatment with the immunecheckpoint inhibitor nivolumab (RCI;n¼ 2), were analyzed with regard to Tcells in duplicates and triplicates.A, Pregating for purified CD4þ andCD8þ T cells from viable (viaþ) CD45þ

CD3þ cells for stimulation experimentsat baseline. B and C, CD8þ andCD4þ T cells of RC- and RCI-treatedpatients were analyzed at baselinefor PD-1 and TIM-3 expression.D, CD8þ T cells of RC- and RCI-treatedpatients were stimulated for 24 and 72hours with anti-CD3, anti-CD28, andVEGF as indicated. Dot plots refer torepresentative RCI and show PD-1 andTIM-3 expression of CD8þ T cells afterstimulation. E and F, Fold change ofPD-1/TIM-3 double-positive fraction ofCD8þ T cells was analyzed. Valueswere normalized to baseline.Statistical analysis was done usingthe Student t test (ns, not significant;� , P < 0.05; �� , P < 0.01; ��� , P < 0.001;error bars, SEM).

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PD-1/TIM-3 double-positive CD8þ T cells was significantlyincreased after 24 hours of VEGF costimulation of peripheralblood T cells from patients who progressed after an initialresponse to nivolumab (Fig. 4D–F; Supplementary Fig. S15). Wefound a similar, but not as prominent, effect in CD8þ T cells frompatients treated with RC. However, comparing both patientcohorts, the fraction of PD-1/TIM-3 double-positive CD8þ T cellswas more markedly increased in the RCI cohort (Fig. 4E and F).Similar results were obtained for CD4þ T cells (SupplementaryFig. S16).

We further analyzed activationmarkers on T cells isolated fromlung, spleen, and blood of mice bearing SCLC. Interestingly, theexpression of CD44 and CD69 on splenic T cells mimicked theactivation pattern of T cells isolated from lungs harboring mac-roscopic tumors (Supplementary Fig. S17). In T-cell stimulationsfor 24 hours using anti-CD28 and VEGF, we observed increasedfractions of splenic T cells expressing PD-1 and TIM-3 (Supple-mentary Fig. S18).

DiscussionOur study demonstrates that combined inhibition of VEGF and

PD-L1 improves PFS and OS in an autochthonous mouse modelof SCLC in a synergistic manner. We observed limited and short-term response to anti–PD-L1 monotherapy in mice with SCLC,which is in accordance with the limited efficacy of anti–PD-1treatment in patients with SCLC (37). Furthermore, we identifiedupregulated expression of the negative regulatory exhaustionmarkers TIM-3 and PD-1 on CD8þ and CD4þ T cells of SCLCsthat acquired resistance to PD-1/PD-L1 blockade in mice andpatients. We reproduced in vitro that this TIM-3–associatedexhausted phenotype is regulated by VEGF signaling in CD4þ

and CD8þ T cells.TIM-3 has been described as a T-cell exhaustion marker that

is coexpressed with PD-1 upon failure of antimicrobial andantitumor responses (38, 39). In line with our data, upregula-tion of TIM-3 upon resistance to PD-1/PD-L1 blockade hasbeen described in colorectal cancer (23), head and neck cancer(40), NSCLC (41), and melanoma (42). However, sequentialTIM-3 blockade overcame acquired resistance against anti–PD-1 therapy only for short term in a murine autochthonousNSCLC model (41).

Recent reports described a VEGF/VEGFR-mediated expres-sion of TIM-3 upon resistance to PD-1 blockade (23, 40). Inline with these findings, we found that the PD-1/TIM-3–exhausted T-cell phenotype is regulated by VEGF and rescuedupon combined anti-VEGF/anti–PD-L1 treatment. Most strik-ingly, combined inhibition of VEGF and PD-L1 synergisticallyimproves OS in mice with SCLC.

One has to consider that in the majority of patients with SCLC,tumors are induced by heavy and extended smoking (43). There-fore, SCLC in patients likely harbor an increased mutational loadwith higher immunogenicity compared with lung carcinomasoccurring in autochthonous mouse models (44, 45). For thisreason, patients with SCLC are probably more amendable toimmunotherapies.

Allen and colleagues showed that combined antiangiogenic/anti–PD-L1 treatment facilitates the activation and infiltration ofT cells into the tumor tissue (46).Weobserved an improvedCD4þ

T-cell infiltration in SCLCs of mice that received combined anti-VEGF/anti–PD-L1 therapy. In line with our data, in patients with

renal cell carcinoma treated with atezolizumab (anti–PD-L1) andbevazizumab (anti-VEGF), a massive infiltration of cytotoxic Tcells was found, as well (47).

Strikingly, Gordon and colleagues identified prolonged sur-vival in tumor mouse models due to increase macrophagedependent antitumor immune responses and phagocytosismediated by PD-1 blockade on TAMs (35). We found a signif-icant upregulation of LAG-3 and CTLA-4 on TAMs upon resis-tance to PD-L1 treatment, which is rescued by combined VEGF/PD-L1 blockade. This might indicate that combined anti-VEGF/anti–PD-L1 therapy abrogates exhaustion of TAMs triggeringprolonged survival of mice with SCLC.

We further observed that galectin-9 was upregulated in TAMsupon acquired resistance against PD-L1 blockade in mice andcoexpressed with TIM-3 in patients with SCLC. In line with ourfindings, elevated galectin-9 expression had been detected inacquired resistance against PD-1 blockade in NSCLC (41) andis known tomediate apoptosis of CD4þ andCD8þ T cells via TIM-3 (33, 48). These findings indicate that galectin-9 expression onTAMs might contribute to T-cell exhaustion.

Upon resistance to immune checkpoint-targeted therapy, Tcells become exhausted and lose their effector functions and theexpression of TNFa and IFNg (38, 40). We did not detect differ-ential IFNy expression among the different therapy groups. How-ever, we found a significantly increased fraction of Tregs uponresistance to combined anti-VEGF/anti–PD-L1 therapy. Otheralternative resistance mechanisms to VEGF blockade might beinitiated by macrophages that were attracted toward the tumorand generate an immunosuppressive tumor microenvironment(49, 50) or by incomplete DC maturation (18). Thereby tumorcell–derived VEGF abrogates expression of immune stimulatorymolecules such as CD80, CD86, and MHC class II and thus DCmaturation by interfering with the NFkB pathway (18, 51).Moreover, combined antiangiogenic/anti–PD-L1 treatment hasbeen described to facilitate the activation and infiltration of DCsand T cells into the tumor tissue (46). However, we did not detectincreased fractions of DCs associated with SCLCs treated withcombined anti-VEGF/anti–PD-L1 therapy.

In summary, we identified an exhausted T-cell phenotypeindicated by PD-1 and TIM-3 expression as a likely adaptiveresistance mechanism to PD-1/PD-L1 blockade in mice andpatients with SCLC. We show that the expression of the immu-nosuppressive receptor TIM-3 on tumor-educated T cells is reg-ulated by VEGF signaling.

Strikingly, combined blockade of VEGF and PD-L1 results insynergistic treatment effects in an autochthonousmousemodel ofSCLC. These results strongly recommend simultaneous VEGF-and PD-L1 inhibition as a therapeutic strategy for the treatment ofpatients with SCLC.

Disclosure of Potential Conflicts of InterestR. Buettner is a consultant/advisory board member for BMS, MSD, Roche,

Pfizer, Qiagen, AbbVie, and AstraZeneca. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: H.A. Schl€oßer, R. Buettner, J. Wolf, M. von Bergwelt-Baildon, R.T. UllrichDevelopment ofmethodology: L.Meder, P. Schuldt, F. Dietlein, S. Borchmann,I. Vlasic, S. Oberbeck, K. Golfmann, M. Herling, H.C. Reinhardt, R.T. UllrichAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Meder, P. Schuldt, M. Thelen, A. Schmitt, S. Klein,

Combined VEGF/PD-L1 Blockade in an SCLC Mouse Model

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K. Wennhold, I. Vlasic, S. Oberbeck, R. Riedel, K. Golfmann, H.A. Schl€oßer,R. Buettner, J. Wolf, M. Herling, R.T. UllrichAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Meder, P. Schuldt, M. Thelen, F. Dietlein, S. Klein,S. Borchmann, K. Wennhold, H.A. Schl€oßer, R. Buettner, J. Wolf, M. Hallek,M. von Bergwelt-Baildon, R.T. UllrichWriting, review, and/or revision of the manuscript: L. Meder, M. Thelen,F. Dietlein, S. Borchmann, K. Wennhold, H.A. Schl€oßer, R. Buettner, J. Wolf,M. Hallek, M. Herling, M. von Bergwelt-Baildon, H.C. Reinhardt, R.T. UllrichAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Dietlein, S. Klein, A. Florin, M. Odenthal,H.C. Reinhardt, R.T. UllrichStudy supervision: R.T. Ullrich

AcknowledgmentsThis work was supported by the Deutsche Krebshilfe (grant no. 70113009,

to R.T. Ullrich), the Thyssen Foundation (grant no. 10.16.1.028MN toR.T. Ullrich), the Nachwuchsforschungsgruppen-NRW (grant no. 1411ng005

to R.T. Ullrich), the Deutsche Forschungsgemeinschaft (DFG; grant no. UL379/1-1 to R.T. Ullrich and KFO-286 RP2/CP1 to H.C. Reinhardt), the Volkswa-genstiftung (Lichtenberg Program; to H.C. Reinhardt), the Bundesminister-ium fu€ur Bildung und Forschung as part of the e:Med program(grant no. SMOOSE 01ZX1303A to H.C. Reinhardt), the German federalstate North Rhine Westphalia (NRW) as part of the EFRE initiative (grant no.LS-1-1-030a to H.C. Reinhardt), the Else Kr€oner- Fresenius Stiftung (grantno. EKFS-2014-A06 to H.C. Reinhardt), the Deutsche Krebshilfe (grant no.111724 to H.C. Reinhardt), and the Center for Molecular Medicine Cologne(CMMC; to R. B€uttner, H.C. Reinhardt, and M. Odenthal).

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.

Received July 19, 2017; revised February 23, 2018; accepted May 15, 2018;published first May 18, 2018.

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Combined VEGF/PD-L1 Blockade in an SCLC Mouse Model

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