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Title

Combined VEGF and PD-L1 blockade displays synergistic treatment effects in an autochthonous mouse

model of small cell lung cancer

Authors

Lydia 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. Schlößer3,9, Margarete Odenthal2,6, Reinhard

Buettner2,6,10, Juergen Wolf1,10, Michael Hallek1,10, Marco Herling1,10, Michael von Bergwelt-Baildon1,3,10, H.

Christian Reinhardt1,10, Roland T. Ullrich1,2,10*

Affiliations

1 Department I of Internal Medicine, University Hospital Cologne, Kerpener Straße 62, 50937 Cologne,

Germany

2 Center for Molecular Medicine Cologne, University of Cologne, Robert-Koch Straße 21, 50931

Cologne, Germany

3 Cologne Interventional Immunology, University Hospital Cologne, Kerpener Straße 62, 50937 Cologne,

Germany

4 Department of Medical Oncology, Dana-Faber Cancer Institute, Boston, Massachusetts, 02215, US

5 Cancer Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142, US

Institute for Pathology, University Hospital Cologne, Kerpener Straße 62, 50937 Cologne, Germany

6 Institute for Pathology, University Hospital Cologne, Kerpener Straße 62, 50937 Cologne, Germany

7 Else Kröner Forschungskolleg Clonal Evolution in Cancer, University Hospital Cologne, Weyertal 115b,

50931, Cologne, Germany

on July 8, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 18, 2018; DOI: 10.1158/0008-5472.CAN-17-2176

http://cancerres.aacrjournals.org/

2

8 German Hodgkin Study Group, Department I of Internal Medicine, University Hospital Cologne,

Kerpener Straße 62, 50937 Cologne, Germany

9 Department of General, Visceral and Cancer Surgery, University Hospital Cologne, Kerpener Straße 62,

50937 Cologne, Germany

10 Center for Integrated Oncology Cologne/Bonn, University Hospital Cologne, Kerpener Straße 62, 50937

Cologne, Germany, University Hospital Bonn, Sigmund-Freud Straße 25, 53105 Bonn, Germany

Running title

Combined VEGF/PD-L1 blockade in a SCLC mouse model

Keywords

Immune checkpoint therapy, small cell lung cancer, PD-L1, VEGF, acquired resistance

* Corresponding author

Roland Ullrich, MD, PhD

Clinic I for Internal Medicine

University Hospital Cologne

Kerpener Straße 62, D-50937 Cologne

Phone: +49 221 478-89771, Fax: +49 221 478-32083

Email: [email protected]

Conflict of interest

All authors declare no potential conflict of interest.



mailto:[email protected]


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4,966

Figures

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Abstract

Small cell lung cancer (SCLC) represents the most aggressive pulmonary neoplasm and is often diagnosed

at late stage with limited survival, despite combined chemotherapies. We show in an autochthonous

mouse model of SCLC that combined anti-VEGF/anti-PD-L1 targeted therapy synergistically improves

treatment outcome compared to anti-PD-L1 and anti-VEGF monotherapy. Mice treated with anti-PD-L1

alone relapsed after 3 weeks and were associated with a tumor-associated PD-1/TIM-3 double positive

exhausted T cell phenotype. This exhausted T cell phenotype upon PD-L1 blockade was abrogated by the

addition of anti-VEGF targeted treatment. We confirmed a similar TIM-3 positive T cell phenotype in

PBMC of SCLC patients with adaptive resistance to anti-PD-1 treatment. Mechanistically, we show that

VEGF-A enhances co-expression of the inhibitory receptor TIM-3 on T cells, indicating an

immunosuppressive function of VEGF in SCLC patients during anti-PD-1 targeted treatment. Our data

strongly suggest that a combination of anti-VEGF and anti-PD-L1 therapies can be an effective treatment

strategy in patients with SCLC.

Significance

Combining VEGF and PD-L1 blockade could be of therapeutic benefit to small cell lung cancer patients.




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Introduction

SCLC accounts for 13-18 % of primary lung cancer cases and is the most aggressive form of pulmonary

carcinomas, mostly diagnosed at late stages with systemic metastases. Although current chemotherapies

are initially effective in patients with SCLC, responses are typically transient and patients succumb to their

disease within a few months after diagnosis (1, 2). Therefore, there is a critical need to convert therapy

responses into durable remissions and to improve outcomes in SCLC patients.

Immune checkpoint blockade using monoclonal antibodies targeting CTLA-4, PD-1 and PD-L1 provided

clinical activity in several cancer types including lung cancer (3, 4). Inhibitory immune checkpoint

receptors including also LAG-3, TIM-3 and TIGIT block T cell effector functions and thereby the elimination

of tumor cells (5) and its expression has been described as prognostic factor in patients with SCLC

(Abstract 8569, ASCO 2017). The limitation of immune checkpoint blockade in cancer therapy is the

activation of different immunosuppressive mechanisms in the tumor microenvironment which abrogate T

cell effector functions and inhibit the infiltration of tumor-educated T cells into the tumor (6). Since

crosstalk between the tumor immune microenvironment and the tumor vasculature contribute to tumor

immune evasion, combined therapy regimens targeting additional immune and/or vascular factors may

provide sustained and potent anti-tumor immune responses (6).

As examples of dual immune checkpoint targeting, in advance melanomas PD-1 blockade provided an

overall response rate of 33 % (7) whereas combined PD-1/CTLA-4 blockade revealed 72 % ORR but with

frequent adverse immune-related toxicities (8). In SCLC, treatment with nivolumab (anti-PD-1) alone

provided ORR of 11 % and in combination with ipilimumab (anti-CTLA-4) ORR of 25 % (CheckMate032). In

contrast to non-squamous non-small cell lung cancer (NSCLC) PD-L1 expression on tumor cell membranes

does not predict response to PD-1 targeted therapies in SCLC (9-11). Thus, SCLC stills lacks a prediction

marker for response to PD1/PD-L1 blockade whereby more than 80 % of SCLC did not respond to

nivolumab (11).




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The lack of broad responses to dual immune checkpoint blockade in SCLC (11) might refer to additional

immunosuppressive mechanisms in the tumor microenvironment which are not directly triggered by T

cell effector functions. The infiltration of regulatory T cells (Tregs) (12), myeloid-derived suppressor cells

(MDSCs) (13) and tumor-associated macrophages (TAMs) may reduce the anti-tumor activity of immune

checkpoint blockades and therefore present a valuable target for novel combined therapy approaches (14,

15).

Several studies indicated that anti-VEGF targeted therapy transforms the tumor immune

microenvironment towards an immunosupportive phenotype (16). Dendritic cells (DC) are antigen-

presenting cells that take up antigens and present them to T cells. Recent studies showed that VEGF

suppresses the maturation of DC precursors and that VEGF blockade improved dendritic cell function and

thereby the efficacy of immunotherapy in cancer (17). In addition, high VEGF levels promote the

proliferation of Tregs and the expansion of immature myeloid cells cells which contribute to tumor-

associated immunosuppression by suppressing antigen-specific T-cell responses (16). High intratumoral

VEGF levels lead to an abnormal growth of tumor vessls that are characterized by hyperpermeabel

functionally insufficient vessels. This insufficient perfusion is associated with a hypoperfused, hypoxic

tumor microenvironment with a high interstitial fluid pressure which impedes T-effector cell infiltration

into the tumor and a shift of TAMs towards an immune inhibitory M2-like phenotype with suppressive T-

effector cell function (6). Anti-VEGF treatment rescues the expression of adhesion proteins, such as E-

selectin and ICAM-1, on endothelial cells in the tumor microenvironment and thereby enable effector T

cell migration into the tumor tissue (18-21).

Importantly, blocking VEGF/VEGFR signaling was described to directly regulate the expression of

inhibitory immune checkpoint receptors on tumor educated T cells (22). Recent data showed in patients

with melanoma that reduced VEGF-A levels are associated with response to anti-PD-1 targeted treatment

suggesting that VEGF-A expression might be associated with response to PD-1/PD-L1 blockade (23).




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VEGF-A, VEGF receptors (VEGFRs) and PD-L1 are highly expressed in patients with SCLC (24, 25). Thus, we

investigated the therapeutic efficacy of combined anti-PD-L1 and anti-VEGF targeted therapy in a Cre-

inducible autochthonous mouse model of SCLC (26).

Materials and Methods

Animal Experiments

This study was performed in accordance to FELASA recommendations. The protocol was approved by the

local Ethics Committee of Animal experiments. The genetically engineered mouse model of SCLC is

driven by a Cre-inducible conditional Rb1 and Tp53 knock out with flox out of Exon 2 to 10 in Tp53 and

Exon 19 in Rb1, as previously described (26). Six-to-eight-week-old male and female

C57BL/6JxFVB/NJx129/Sv mice were anesthetized with Ketamin/Xylazin (100 mg/kg/(body weight) BW

i.p./0.5 mg/kg/BW i.p.) and 2.5x10^7 pfu Adeno-Cre was applied intratracheally (27). Viral vectors were

provided by the University of Iowa Viral Vector Core (http://www.medicine.uiowa.edu/vectorcore). An

initial cohort was used to determine survival from the time point of inhalation and estimate a starting

point for monitoring initial tumor growth (Supplementary Fig. S1). Serial µCT to monitor tumor induction

in the therapy groups were started from week 22 after Cre application and target lesions were correctly

identified from isolated lung tissue (Supplementary Fig. S1). For µCT measurements (LaTheta mCT,

Hitachi Alcoa Medical, Ltd), mice were anesthetized using 2.5 % isofluran. Histologically, SCLC primary

tumors resembled human SCLC with regard to cell morphology determined by hematoxylin and eosine

(H&E) stain, proliferation determined by Ki-67 stain and NE marker expression, here CD56. Moreover,

SCLC tumors expressed PD-L1 and VEGF (Supplementary Fig. S1).

Upon a measurable target lesion, mice were randomly distributed into groups (Supplementary Fig. S1,

Supplementary Table S1) and mice were imaged by µCT once a week. SCLC cohorts comprised five

therapy groups and all therapies were given every 3 days simultaneously: (1) vehicle (phosphate buffered



http://www.medicine.uiowa.edu/vectorcore


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saline; PBS); (2) IgG (corresponding monotherapy IgGs (Southern Biotech, US) diluted in PBS) (3) anti-

mouse VEGF-A monoclonal antibody (aVEGF, B20-4.1.1-PHAGE, kindly provided by Genentech) (5

mg/kg/BW i.p.); (4) anti-mouse PD-L1 monoclonal antibody (aPD-L1, clone 6E11, kindly provided by

Genentech) (5 mg/kg/BW i.p.) and (5) combined anti-VEGF/anti-PD-L1 (5 mg/kg/BW/5mg/kg/BW i.p.)

where both compounds were applied simultaneously. Reagents were diluted in PBS and obtained from

Genentech (San Francisco, CA, USA) who specified the therapy regimen. As a reference group, SCLC-

bearing mice were treated with cycles of a standard combined chemotherapy regiment comprising

Cisplatin (5 mg/kg/BW, 1x per week) and Etoposide (10 mg/kg/BW, 3x per week) followed by 2-3 week

recovery time dependent on toxicity and weight loss (Supplemetary Fig. S1). Tumor growth was

monitored by serial µCT whereby the RECIST criteria v1.1 (28) were adapted to the SCLC model. The

minimal measurable target lesion by µCT scans was adapted to 1 mm. Slice thickness was adapted to 0.3

mm. The first dose was given upon target lesion identification and baseline evaluation, maximally one

day before. Response criteria to evaluate the target lesion were maintained with regard to diameter fold

change. Complete response (CR) referred to a decrease of 100 %, partial response (PR) was indicated

upon a >30 % reduction, progressive disease (PD) referred to an increase of >20 % and/or new lung

lesions and stable disease (SD) was termed upon a diameter change that did not qualify for PR or PD. µCT

data was analyzed using OsiriX-DICOM viewer (aycan Digitalsysteme GmbH). PFS and OS of the therapy

groups and the predictive additive probability of survival of both monotherapy-treated cohorts were

analyzed as follows: Let 𝑝𝐴 (𝑡), 𝑝𝐵 (𝑡), 𝑝𝐴,𝐵 (𝑡) and 𝑝Cntrl (𝑡) denote the probability of survival for

𝑡 ∈ [0; ∞) under therapy with compound 𝐴, 𝐵, their combination or vehicle solution, respectively. In

order to determine whether the combination two drugs 𝐴 and 𝐵 had a synergistic impact on survival, we

calculated the expected additive curve as

𝑝𝐴+𝐵(𝑡) ∶= min(1, 𝑝Cntrl(𝑡) + |𝑝𝐴(𝑡)– 𝑝Cntrl(𝑡)|≥0 + |𝑝𝐵(𝑡)– 𝑝Cntrl(𝑡)|≥0)




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where 𝑝𝐴+𝐵(𝑡) denotes the expected probability of survival, assuming the combination effect of drugs 𝐴

and 𝐵 is additive, and |𝑎|≥0 ∶= max (0, 𝑎). Based on this definition, we calculated the expected number

of events at each time point of 𝑝𝐴+𝐵(𝑡) by inverting the Kaplan-Meier statistics, assuming equal cohort

sizes between combination and single-agent cohorts. Based on these events, we finally compared the

expected additive survival rate 𝑝𝐴+𝐵(𝑡) and the observed survival rate 𝑝𝐴,𝐵 (𝑡) under drugs 𝐴 and 𝐵 in

combination, using a Mantel-Cox test. Scripts are available upon request.

Flow Cytometry

Organs of mice were harvested and cells were isolated by mechanical dissociation using 40 µm cell

strainers (BD Falcon). Red blood cells were lysed by ACK lysis buffer (Life Technologies) and cells were

washed with PBS. Purified primary cells and T cells were stained for 30 min at 4 °C for flow cytometry

using antibodies against following targets and isotype controls, both obtained from BioLegend 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), IFNγ (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 (Alexa Fluor 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) and Rat

IgG2aΚ (FITC, PE, PerCP-Cy5.5, APC, Alexa Fluor 700), PE-Dazzle594 Armenian Hamster IgG (PE-

Dazzle594), Rat IgG2bΚ (PE-Cy7) and mouse BALB/c IgG2aΚ (Pacific Blue). In addition, APC-Cy7

conjugated fixable viability dye (eBioscience) or the Zombie Aqua Fixable Viability Kit (BioLegend) was

used.

Purified human T cells were stained in 50 µL volume for 20 min at 4 °C for flow cytometry using

antibodies against following targets, obtained from BioLegend if not otherwise specified: CD45 (FITC,




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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-Fire 750, FN50). In addition, a pacific orange conjugated fixable

viability dye (zombie aqua) was used. Flow cytometry for murine and human cells was performed on a

Gallios 10/3 (Beckman Coulter) and data was analyzed using FlowJo (Tree Star v7.6.1).

T cell Stimulation

Murine T cells were isolated from harvested spleens of mice harboring SCLC using MojoSort Mouse CD3

T cell Isolation (BioLegend) according to manufacturer’s protocol. T cells were cultured in 96-well flat

bottom plates (Sarstedt) and stimulation was performed for 24 h using 5 µg immobilized anti-CD3 (BD

Pharmingen), 2µg/ml soluble anti-CD28 (BD Pharmingen), 10 ng/ml murine IFNγ (PeproTech) and 50

ng/ml murine VEGF165 (PeproTech), also known as VEGF-A. Human peripheral blood mononuclear cells

(PBMCs) were isolated from SCLC patients’ blood between 2 to 4 weeks after the last received dose by

density gradient centrifugation using Pancoll (Pan Biotech, density 1.077 g/l). T cells were purified by

magnetic cell sorting using MojoSort Human CD3 T cell isolation (BioLegend) according to manufacturer’s

protocol for column free isolation. T cells were cultured in 96-well flat bottom plates (Sarstedt) under

humanized conditions and stimulated for 24 h and 72 h using 5 µg/ml immobilized anti-CD3 (BioLegend),

2 µg/ml soluble anti-CD28 (BioLegend) and 50 ng/ml human VEGF165 (PeproTech).

Immunohistochemistry

Murine organs were harvested and fixed in 4 % PBS-buffered formalin for paraffin embedding. 3 µm

tissue sections were deparaffinized and immunohistochemistry (IHC) was performed using the LabVision

Autostainer-480S (Thermo Scientific) staining with hematoxylin & Eosine (H&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, A-20), CD4 (abcam, EPR19514), CD8

(abcam polyclonal, ab203035), FOXP3 (Novus Biologicals, polyclonal, NB100-39002) and the Secondary-




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Histofine-Simple-Stain (SHSS) antibody detection kit (Medac). Slides were scanned by the Panoramic-250

slide scanner (3D Histech). CD31 staining was used to determine microvessel vessel density. Briefly, five

representative, 20x enlarged fields were extracted from each slide using the Panoramic Viewer Software.

A customized script of ImageJ (National Institutes of Health, US) was used to identify CD31 positive

structures. The number of structures with a minimum size of 30 pixels were counted per 20x enlarged

field and averaged across all fields for each slide. Human SCLC was diagnosed based on histological

examination by trained lung-pathologists. Pictures were acquired with a Leica-DM-5500 B Microscope.

Primary antibodies against PD-L1 (28-8, abcam), TIM-3 (D5D5R, Cell Sinaling), Galectin-9 (D9R4A, Cell

Signaling), CD56 (123C3, Zytomed), Synaptophysin (SP11, Thermo Fisher) and Chromogranin A (DAK-

A3,Dako) were used. Secondary antibodies were purchased from ImmunoLogic (BrightVision+) and

staining was performed using the LabVision Autostainer 480S (Thermo Scientific).

Ethics

All human subject research was performed in strict accordance with approved protocols by the local

ethics committee of the University Hospital Cologne and with the recognized ethical guidelines of the

Declaration of Helsinki. Blood samples (reference number 17-130) and tumor tissue (reference number

10-242) were obtained during routine clinical procedures from patients diagnosed based on the World

Health Organization classification of lung tumors (29) providing written informed consent for additional

tissue collection.

Statistical Tests

Statistics were done using Prism (GraphPad V5.0) and SPSS (IBM, V24.0). Error bars indicated standard

error of the mean (SEM). P-values <0.05 were regarded as significant and indicated in the figures: * P

0.05, **P 0.01, *** P 0.001.




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Results

Combined anti-VEGF/anti-PD-L1 targeted therapy synergistically improves PFS and OS in SCLC

We performed a therapeutic study in an autochthonous mouse model of SCLC in which tumors are

induced upon Cre-mediated bi-allelic deletion of Rb1 and Tp53. We recorded the clinicopathologic

parameters of SCLC bearing mice listed according to the applied therapy regiments (Supplementary

Table S1). Mice were randomized and systemically treated with vehicle, corresponding IgGs, anti-VEGF,

anti-PD-L1 and with the combination of anti-VEGF and anti-PD-L1 targeting. Anti-VEGF monotherapy in

SCLC-bearing mice did not improve progression-free survival (PFS) and overall survival (OS) (1 week, 3

weeks, respectively) in comparison to vehicle-treated mice (1 week, 3 weeks, respectively) and IgG

treated mice (1.5 weeks, 2.5 weeks, respectively) (Fig. 1A and B). Treatment with anti-PD-L1 alone

significantly improved PFS (2 weeks, P=0.0061) and OS (4 weeks, P=0.0008) compared to the vehicle

group. Most strikingly, combined anti-VEGF/anti-PD-L1 inhibition led to a substantial improvement in

median PFS (3 weeks) and OS (6 weeks) in comparison to anti-PD-L1 monotherapy (PFS: P=0.0166, OS:

P=0.0231) (Supplementary Tables S2-S5). To decipher whether the combination of anti-VEGF/anti-PD-L1

targeted therapy results in synergistic treatment effects we calculated predicted additive PFS and OS

curves as described in the Materials and Methods. Using Prism Mantel-Cox test (Supplementary Table S2

and S3) we compared the predicted additive curves of OS and PFS with the observed survival curves of

the anti-VEGF/anti PD-L1 combined therapy group (PFS: 0.0119, OS: P=0.0316) (Fig. 1C and D). Since we

determined a significant difference between the predicted additive and the combination therapy curves,

the therapeutic effect on survival of anti-VEGF and anti-PD-L1 targeted therapy which were administered

simultaneously, was defined as synergistic effect. We further compared OS data of combined anti-

VEGF/anti-PD-L1 treatment with standard combined Cisplatin/Etoposide chemotherapy. Of note, OS of

the mice with SCLC treated with combined anti-VEGF/anti-PD-L1 therapy was better than the observed




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OS upon standard combined chemotherapy regiments (median OS: 5 weeks, 6 weeks, respectively,

P=0.1312) (Supplementary Fig. S1).

We next assessed the change in target lesion diameter in all therapy groups after 1 and 2 weeks of

treatment using µCT analysis. After 1 week of treatment we found one stable disease (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

partial response (PR) was found in 7.1 % of mice (1 of 14) treated with anti-PD-L1 and 7.7 % of mice (1 of

13) treated with combined anti-VEGF/anti-PD-L1. However, already after one week, 80.0 % of mice (8 of

10) treated with anti-VEGF monotherapy, 50 % of mice (3 of 6) treated with IgG controls and 100% of

vehicle-treated mice (12 of 12) showed progressive disease (PD) (Fig. 1E). After 2 weeks of treatment,

stable disease (SD) was detected upon anti-PD-L1 monotherapy in 42.9 % (6 of 14) and upon combined

anti-VEGF/anti-PD-L1 in 46.2 % (6 of 13). Of note, a partial response (PR) was only determined in SCLC-

bearing mice treated with combined anti-VEGF/anti-PD-L1 (15.4 %; 2 of 13). PD was determined in all

vehicle-treated (12 of 12), all IgG-treated (6 of 6) and all of the anti-VEGF-treated mice (10 of 10). Of

anti-PD-L1 monotherapy-treated mice, 42.9 % (6 of 14) showed a PD, while only 23.1 % (3 of 13) of

combined anti-VEGF/anti-PD-L1-treated mice showed progressive tumors (Fig. 1F). Representative serial

µCT measurements indicated a PR upon combined anti-VEGF/anti-PD-L1 therapy followed by a SD at 4

weeks and a final PD after 7 weeks of treatment (Fig. 1G).

Taken together, treatment of SCLC-bearing mice with combined anti-VEGF and anti-PD-L1 targeted

therapy synergistically improves PFS and OS compared to anti-PD-L1 and anti-VEGF alone.

Anti-PD-L1 therapy induces an exhausted T cell phenotype that is diminished in mice with combined anti-

VEGF and anti-PD-L1 treatment




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We next analyzed the impact of anti-PD-L1, anti-VEGF and combined anti-VEGF/anti-PD-L1 treatment on

tumor infiltrating T cells. First, the localization of CD4+, CD8+ and FOXP3+ T cells was examined using IHC

(Figure 2A). In vehicle treated mice with SCLC, T cells did not accumulate in the pulmonary tissue around

the tumor and did not infiltrate the tumor tissue. In anti-VEGF treated mice, tumors were infiltrated by

few CD4+ T cells, whereby CD8+ and FOXP3+ T cells remained at the tumor margin. In anti-PD-L1 treated

mice, CD4+ and FOXP3+ T cells accumulated in the pulmonary tissue at the tumor margin but did not

invade tumor tissue. In SCLC-bearing mice treated with combined anti-VEGF/anti-PD-L1 CD4+ T cells and

few FOXP3+ and CD8+ T cells infiltrated tumor tissue. We also calculated CD31 positive tumor

microvessels in progressed SCLC lesions upon therapy and did not detect significant differences in

microvessel density (Supplementary Fig. S2).

Second, we generated single cell suspensions from primary tumors and immuno-detected tumor cells

and immune cells, including T cells (CD45+ CD3+). We found a significantly increased ratio of pan-

immune cells (CD45+) to tumor cells (CD45- CD56+) but the fraction of T cells within the immune cell

compartment. CD4/CD8 ratio and IFNγ expression were not significantly altered upon anti-PD-L1 and

combined anti-VEGF/anti-PD-L1 targeted therapy compared to vehicle (Supplementary Fig. S3,

Supplementary Fig. S4). However, we found a significant increase in the fraction of Tregs (CD4+ FOXP3+)

in tumors that progressed upon combined anti-VEGF/anti-PD-L1 therapy (Supplementary Fig. S5). We

further analyzed the fractions of Thelper1 (Th1) and Th2 CD4+ T cells using CXCR3 and CCR4 markers.

The ratio of Th1 (CD4+ FOXP3- CXCR3+) and Th2 (CD4+ FOXP3- CCR4+) cells was not significantly affected

using anti-VEGF and anti-PD-L1 targeted therapies (Supplementary Fig. S5).

With regard to CD8+ T cells, their assembly at the site of the tumor was significantly increased upon the

initial response to combined anti-VEGF/anti-PD-L1 treatment (Supplementary Fig. S6) which disappeared

upon progressive disease.




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To elucidate mechanisms of adaptive resistance, we analyzed immune checkpoint expression in tumor-

infiltrating lymphocytes. Generally, the T cell function is mediated by receptor-ligand interactions, which

may have stimulatory or inhibitory effects. An exhausted T cell phenotype is indicated by simultaneous

upregulation of at least two inhibitory receptors, such as PD-1 and T cell immunoglobulin mucin-3 (TIM-

3) (3). Herein, we found upregulation of the immune checkpoints TIM-3, LAG-3 and PD-1 on CD4+ and

CD8+ T cells in tumors with adaptive acquired resistance against anti-PD-L1 therapy (PD-1/TIM3: CD4+

P=0.0081 and CD8+ P=0.0071; PD-1/LAG-3: CD4 P=0.0082 and CD8 P=0.0395) (Fig. 2B-E). CTLA-4 and PD-

1 double positive T cell fractions were not increased upon acquired anti-PD-L1 resistance

(Supplementary Fig. S7). This exhausted T cell phenotype was significantly increased in tumors that

progressed during anti-PD-L1 treatment in comparison to anti-PD-L1 treated mice with partial response

and stable disease which represents a true phenotype acquisition (CD4+ P=0.0310 and CD8+ P=0.0062;

Supplementary Fig. S6). Moreover, we found that the exhausted T cell phenotype was locally associated

to the presence of tumor cells (Supplementary Fig. S8).

Interestingly, the PD-1/TIM-3 exhausted T cell phenotype was significantly rescued by combining anti-

PD-L1-targeted therapy with anti-VEGF therapy (CD4+ P=0.0389; CD8+ P=0.0411, respectively), whereas

the PD-1/LAG-3 exhausted phenotype was not rescued (Fig. 2B-E). We also show that the exhausted T

cell phenotype was not dependent on IFNγ (Supplementary Fig. S9). Moreover, VEGF knock-out in SCLC

tumors did not enhance response to aPD-L1 treatment (Supplementary Fig. 10).

As TIM-3 was upregulated upon progression during anti-PD-L1 targeted treatment which was again

abrogated by the addition of anti-VEGF therapy we hypothesized that VEGF/VEGFR-signaling induce TIM-

3 expression on tumor-associated T cells. We found that VEGFR1 was up-regulated on tumor-associated

CD8+ T cells (Supplementary Fig. 11). Thus, these data might indicate that TIM-3 expression is regulated

by VEGF-VEGFR1 in CD8 T-cells. However, the VEGF induced signaling pathway that regulates TIM-3

expression in CD-8 cells remains elusive.




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Taken together, combining anti-VEGF to anti-PD-L1 targeted therapy rescued T cell exhaustion which was

observed as an acquired resistance mechanism to PD-L1 blockade in SCLC.

Increased Galectin-9 expression in tumor-associated macrophages upon PD-L1 treatment

We further investigated the expression of PD-L1 (30), PD-L2 (31) and Galectin-9 (32) in the tumor

microenvironment. They represent prominent ligands for PD-1 and TIM-3, respectively, and trigger

immunotolerance and tumor immune evasion through the abrogation of IFNγ signaling and the induction

of effector T cell apoptosis (30-32).

We analyzed the expression of these ligands on tumor-associated dendritic cells (TADCs) (CD45+ CD56-

F4/80- CD11c+), tumor cells (CD45- CD56+) and tumor-associated macrophages (TAMs) (CD45+ CD56-

F4/80+). The expression of Galectin-9 was significantly increased on TAMs in SCLCs that progressed

during anti-PD-L1 and combined anti-VEGF/anti-PD-L1 therapy (measured by mean fluorescence

intensity compared to vehicle, anti-PD-L1: P=0.0069, anti-PD-L1/anti-VEGF: P=0.0212, respectively), but

was not significantly altered on TADCs and tumor cells (Fig. 3A and B, Supplementary Fig. S12). In human

SCLC patient samples without anti-PD1/anti-PD-L1 treatment, Galectin-9 was detected on tumor cells (3

of 18), TAMs and lymphocytes (8 of 18) and frequently co-expressed with TIM-3 (7 of 8) but not with PD-

L1 (Supplementary Fig. S13; Supplementary Tables S6 and S7). PD-L1, which was shown to be

significantly expressed on tumor cells and within the tumor microenvironment of SCLC (25), was

significantly reduced on TADCs (anti-PD-L1: P>0.001, anti-PD-L1/anti-VEGF: P=0.002, respectively), tumor

cells (anti-PD-L1: P=0.0111, anti-PD-L1/anti-VEGF: P=0.0220, respectively) and TAMs (anti-PDL1:

P=0.0002, anti-PD-L1/anti-VEGF: P=0.0009, respectively), upon anti-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 low levels on tumor cells. However, PD-L2 was not differentially expressed in

SCLC following the applied therapy regiments (Supplementary Fig. S12). Furthermore, we analyzed the




17

expression of MHC class I on TADCs, TAMs and tumor cells, as genomic aberrations in B2M resulting in

MHC class I loss were identified as potential resistance mechanism to PD-1 blockade in melanoma (33).

However, MHC class I was not differentially expressed between any of the cell types in our four SCLC

therapy cohorts (Supplementary Fig. S12). We also analyzed the ratio of TAMs and TADCs within the

immune cells but did not identify significant alterations upon application of the different therapy

regiments (Supplementary Fig. S3).

Since immune checkpoint receptors were recently discovered on TAMs, abrogating their anti-tumor

function and directly contributing to the response to immune checkpoint blockade (34, 35), 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 expression was induced on TAMs

upon acquired resistance to PD-L1 blockade (PD-1 P<0.0001; TIM3 P=0.1053; LAG-3 P=0.0064; CTLA-4

P=0.0015). Combining anti-PD-L1/anti-VEGF targeted therapy reduced LAG-3 and CTLA-4 expression

significantly (P=0.0012; P=0.0003, respectively) compared to anti-PD-L1 monotherapy. Taken together,

TAMs might present an exhausted phenotype like T cells upon acquired resistance to PD-L1 blockade and

may contribute to the prolonged survival of mice with SCLC achieved by combined anti-VEGF/anti-PD-L1

treatment.

VEGF significantly upregulates TIM-3 on CD8+ T cells isolated from human PBMCs upon acquired

resistance to Nivolumab

To validate our preclinical findings in human patient samples, we isolated PBMCs from patients with

SCLC that progressed during Nivolumab (anti-PD-1) treatment. We investigated PBMCs of two cohorts of

patients with SCLC: those treated with irradiation and chemotherapy alone (RC) or those treated with RC

followed by the immune checkpoint inhibitor nivolumab (RCI). Confirming our preclinical data derived

from mice with autochthonous SCLC, TIM-3 was significantly upregulated on CD8+ and CD4+ T cells of




18

peripheral blood of patients with SCLC that progressed following response to anti-PD-1 therapy. As

expected, PD-1 on these peripheral blood T cells was downregulated due to anti-PD-1 treatment (Fig. 4A-

C).

Following our postulate that VEGF induces TIM-3 expression in tumor-associated T cells, we investigated

the effect of VEGF stimulation on T cells derived from PBMCs of the above-mentioned patient cohorts. In

line with our hypothesis, the fraction of PD-1/TIM-3 double positive CD8+ T cells was significantly

increased after 24 h of VEGF co-stimulation of peripheral blood T cells from patients that progressed

after an initial response to nivolumab (Fig. 4D-F, Supplementary Fig. S15). We found a similar, but not as

prominent, effect in CD8+ T cells from patients treated with RC. However, comparing both patient

cohorts, the fraction of PD-1/TIM-3 double positive CD8+ T cells was more markedly increased in the RCI

cohort (Fig. 4E and F). Similar results were obtained for CD4+ T cells (Supplementary Fig. S16).

We further analyzed activation markers on T cells isolated from lung, spleen and blood of mice bearing

SCLC. Interestingly, the expression of CD44 and CD69 on splenic T cells mimicked the activation pattern

of T cells isolated from lungs harboring macroscopic tumors (Supplementary Fig. S17). In T cell

stimulations for 24 h using anti-CD28 and VEGF, we observed increased fractions of splenic T cells

expressing PD-1 and TIM-3 (Supplementary Fig. S18).




19

Discussion

Our study demonstrates that combined inhibition of VEGF and PD-L1 improves PFS and OS in an

autochthonous mouse model of 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 to the limited efficacy of

anti-PD-1 treatment in patients with SCLC (36). Furthermore, we identified upregulated expression of the

negative regulatory exhaustion markers TIM-3 and PD-1 on CD8+ and CD4+ T cells of SCLCs that acquired

resistance to PD-1/PD-L1 blockade in mice and patients. We reproduced in vitro that this TIM-3

associated exhausted phenotype is regulated by VEGF signaling in CD4+ and CD8+ T cells.

TIM-3 has been described as a T cell exhaustion marker that is co-expressed with PD-1 upon failure of

anti-microbial and anti-tumor responses (37, 38). In line with our data, up-regulation of TIM-3 upon

resistance to PD1/PD-L1 blockade has been described in colorectal cancer (22), head and neck cancer

(39), NSCLC (40) and melanoma (41). However, sequential TIM-3 blockade overcame acquired resistance

against anti-PD-1 therapy only short-term in a murine autochthonous NSCLC model (40).

Recent reports described a VEGF/VEGFR mediated expression of TIM-3 upon resistance to PD-1 blockade

(22, 39). In line with these findings we found that the PD-1/TIM-3 exhausted T cell phenotype is

regulated by VEGF and rescued upon combined anti-VEGF/anti-PD-L1 treatment. Most strikingly,

combined inhibition of VEGF and PD-L1 synergistically improves OS in mice with SCLC.

One has to consider that in the majority of SCLC patients tumors are induced by heavy and extended

smoking (42). Therefore, SCLC in patients likely harbor an increased mutational load with higher

immunogenicity compared to lung carcinomas occurring in autochthoneous mouse models (43, 44). For

this reason, patients with SCLC are probably more amendable to immunotherapies.

Allen and colleagues showed that combined anti-angiogenic/anti-PD-L1 treatment facilitates the

activation and infiltration of T cells into the tumor tissue (45). We observed an improved CD4+ T cell

infiltration in SCLCs of mice which received combined anti-VEGF/anti-PD-L1 therapy. In line with our data,




20

in patients with renal cell carcinoma treated with atezolizumab (anti-PD-L1) and bevazizumab (anti-

VEGF) a massive infiltration of cytotoxic T cells was found, as well (46).

Strikingly, Gordon and colleagues identified prolonged survival in tumor mouse models due to increase

macrophage dependent anti-tumor immune responses and phagocytosis mediated by PD-1 blockade on

TAMs (34). We found a significant up-regulation of LAG-3 and CTLA-4 on TAMs upon resistance 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 triggering prolonged survival of mice with SCLC.

We further observed that Galectin-9 was upregulated in TAMs upon acquired resistance against PD-L1

blockade in mice and co-expressed with TIM-3 in SCLC patients. In line with our findings, elevated

Galectin-9 expression had been detected in acquired resistance against PD-1 blockade in NSCLC (40) and

is known to mediate apoptosis of CD4+ and CD8+ T cells via TIM-3 (32, 47). These findigs indicate that

Galectin-9 expression on TAMs might contribute to T-cell exhaustion.

Upon resistance to immune checkpoint targeted therapy, T cells become exhausted, loose their effector

functions and the expression of TNFα and IFNγ (37, 39). We did not detect differential IFNy expression

among the different therapy groups. However, we found a significantly increased fraction of Tregs upon

resistance to combined anti-VEGF/anti-PD-L1 therapy. Other alternative resistance mechanisms to VEGF

blockade might be initiated by macrophages which were attracted towards the tumor and generate an

immunosuppressive tumor microenvironment (48, 49) or by incomplete DC maturation (17). Thereby

tumor cell-derived VEGF abrogates expression of immune stimulatory molecules such as CD80, CD86 and

MHC class II and thus DC maturation by interfering with the NFkB pathway (17, 50). Moreover, combined

anti-angiogenic/anti-PD-L1 treatment has been describe to facilitate the activation and infiltration of DCs

and T-cells into the tumor tissue (45). However, we did not detect increased fractions of DCs associated

to SCLCs treated with combined anti-VEGF/anti-PD-L1 therapy.




21

In summary, we identified an exhausted T cell phenotype indicated by PD-1 and TIM-3 expression as a

likely adaptive resistance mechanism to PD-1/PD-L1 blockade in mice and patients with SCLC. We show

that the expression of the immunosuppressive receptor TIM-3 on tumor-educated T cells is regulated by

VEGF signaling.

Strikingly, combined blockade of VEGF and PD-L1 results in synergistic treatment effects in an

autochthonous mouse model of SCLC. These results strongly recommend simultaneous VEGF- and PD-L1-

inhibition as a therapeutic strategy for the treatment of patients with SCLC.

Acknowledgements

This work was supported by the Deutsche Krebshilfe (Grant No.: 70113009 to R.T. Ullrich), by the

Thyssen foundation (Grant No.: 10.16.1.028MN to R.T. Ullrich), by 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), by the Volkswagenstiftung

(Lichtenberg Program, H.C. Reinhardt.), by the Bundesministerium fur Bildung und Forschung as part of

the e:Med program (to H.C. Reinhardt, Grant No. SMOOSE 01ZX1303A), by the German federal state

North Rhine Westphalia (NRW) as part of the EFRE initiative (Grant No.: LS-1-1-030a, H.C. Reinhardt), by

the Else Kröner- Fresenius Stiftung (Grant No.: EKFS-2014-A06, H.C.Reinhardt), by the Deutsche

Krebshilfe (Grant No.: 111724 to H.C.Reinhardt) and by the Center for Molecular Medicine Cologne

(CMMC) (to R. Büttner, H.C. Reinhardt and M. Odenthal).




22

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Figure Legends

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=12), IgG control (violet, n=6) anti-VEGF

monotherapy (aVEGF, orange, n=10), anti-PD-L1 monotherapy (aPD-L1, green, n=14) and combined anti-

VEGF/anti-PD-L1 therapy (combi, red, n=13) and serially imaged by µCT. (A and B) Progression free

survival (PFS) and overall survival (OS) were determined from the five therapy groups. Statistical analysis

was done using the Prism Mantel-Cox test (ns – not significant; * P <0.05; ** P <0.01; *** P <0.001; black

star – compared to vehicle; violet star – compared to IgG, orange star – compared to aVEGF; green star –

compared to aPD-L1, blue star – compared to additive). The corresponding P-Values, the Chi-squared value

and the Hazard ratios 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-VEGF and 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. Chi-Squared values and

Hazard ratios ratios are listed in Supplementary Tables S2-S5. (E and F) Change in target lesion diameter

calculated from all therapy groups after 1 week (E) and after 2 weeks (F) of treatment. Striped columns

refer to animals which died before 2 weeks of treatment so the last determined value was plotted. PD –

progressed disease; SD – stable disease; PR – partial response; CR – complete response according to

described mouse adapted RECIST v1.1 criteria. (G) Serial µCT measurements of one representative

mouse per therapy group. Target lesion diameter is marked green. H – heart; D – Diaphragm; † –dead.




29

Figure 2. Anti-PD-L1 resistant SCLC show significantly increased PD-1/TIM-3 double positive CD8+ and

CD4+ T cells.

SCLC-bearing mice were treated with vehicle (n=15), anti-VEGF monotherapy (aVEGF, n=10), anti-PD-L1

monotherapy (aPD-L1, n=11) and combined anti-VEGF/anti-PD-L1 therapy (combi, n=10) and end point

analysis was performed using IHC and flow cytometry. (A) CD4, CD8 and FOXP3 stains on FFPE SCLC

tissue by IHC. Images were taken at 20x magnification. Bars indicate 100µm. (B) CD45+ CD3+ CD8+ T cells

were analyzed for PD-1 and TIM-3 expression. Dot plots of one representative experiment per therapy

group are shown. (C) CD45+ CD3+ CD4+ T cells were analyzed for PD-1 and TIM-3 expression. Dot plots of

one representative experiment per therapy group are shown. (D) CD45+ CD3+ CD8+ T cells were

analyzed for PD-1 and LAG-3 expression. Dot plots of one representative experiment per therapy group

are shown. (E) CD45+ CD3+ CD4+ T cells were analyzed for PD-1 and LAG-3 expression. Dot plots of one

representative experiment per therapy group are shown. Statistical analysis was done using Student’s t-

test (ns – not significant; * P <0.05, error bars indicate SEM).

Figure 3. Anti-PD-L1 resistant SCLC show higher Galectin-9 expression in tumor-associated

macrophages.

SCLC-bearing mice were treated with vehicle (n=4), anti-VEGF monotherapy (aVEGF, n=4), anti-PD-L1

monotherapy (aPD-L1, n=5) and combined anti-VEGF/anti-PD-L1 therapy (combi, n=4). Upon detection of

progressive disease based on µCT measurements and mouse adapted RECIST v1.1 criteria, end point

analysis was performed using flow cytometry. (A) Lysates from primary tumor material were stained for

viable (via+) immune cells (CD45+) and non-immune cells (CD45-) which were used to identify CD56+

SCLC cells. T cells (CD3+) were identified within the CD45+ gate. The CD45+ CD3- cells were used to

identify tumor-associated macrophages (TAMs) using anti-F4/80 and anti-MHCI. The CD45+ CD3- F4/80-

cells were used to identify tumor-associated dendritic cells by anti-CD11c. (B) Relative Galectin-9




30

expression of TAMs, determined by mean fluorescence intensity (MFI), was normalized to IgG control.

Histograms of one representative experiment per therapy group are shown. Statistical analysis was done

using Student’s t-test (ns – not significant; * P <0.05; ** P <0.01, error bars indicate SEM).

Figure 4. Stimulation with VEGF significantly increases the fraction of PD-1/TIM-3 double positive CD8+

T cells.

PBMCs from SCLC patients who received radio-chemotherapy (RC, n=3) or radio-chemotherapy followed

by treatment with the immune checkpoint inhibitor Nivolumab (RCI, n=2) were analyzed with regard to T

cells in duplicates and triplicates. (A) Pregating for purified CD4+ and CD8+ T cells from viable (via+)

CD45+ CD3+ cells for stimulation experiments at baseline. (B and C) CD8+ and CD4+ T cells of RC- and

RCI-treated patients were analyzed at baseline for PD-1 and TIM-3 expression. (D) CD8+ T cells of RC- and

RCI-treated patients were stimulated for 24 h and 72 h with anti-CD3, anti-CD28 and VEGF as indicated.

Dot plots refer to representative RCI and show PD-1 and TIM-3 expression of CD8+ T cells after

stimulation. (E and F) Fold-change of PD-1/TIM-3 double positive fraction of CD8+ T cells was analyzed.

Values were normalized to baseline. Statistical analysis was done using Student’s t-test (ns – not

significant; * P <0.05; ** P <0.01; *** P <0.001, error bars indicate SEM).




vehicle aVEGF aPD-L1 combi B

C

PD1

LAG

3

PD1

LAG

3

D

E

vehicle aVEGF aPD-L1 combi



PD1

TIM

3

PD1

TIM

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veh

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aV

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Figure 2




Published OnlineFirst May 18, 2018.Cancer Res Lydia Meder, Philipp Schuldt, Martin Thelen, et al. cell lung cancertreatment effects in an autochthonous mouse model of small Combined VEGF and PD-L1 blockade displays synergistic

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