Induction of T-cell immunity overcomes complete resistance …cancerimmunolres.aacrjournals.org/content/canimm/early/2015/02/24/... · improve tumor response rates in melanoma, a

Induction of T-cell immunity overcomes complete resistance to PD-1 and CTLA-4

blockade and improves survival in pancreatic carcinoma

Rafael Winograd1, Katelyn T. Byrne1,6, Rebecca A. Evans1,6, Pamela M. Odorizzi3,

Anders R. L. Meyer5, David L. Bajor1,2,4, Cynthia Clendenin1, Ben Z. Stanger1,2,4,

Emma E. Furth5, E. John Wherry3, and Robert H. Vonderheide1,2,4,*

1Abramson Family Cancer Research Institute 2Abramson Cancer Center 3Department of Microbiology 4Department of Medicine 5Department of Pathology and Laboratory Medicine Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA USA 19104 6equal contribution *Correspondence: Robert H. Vonderheide, MD, DPhil

Abramson Family Cancer Research Institute Perelman School of Medicine, University of Pennsylvania 8-121 TRC, Building 421 3400 Civic Center Blvd Philadelphia, PA 19104 Phone: 215-573-4265 Fax : 215-573-2652 Email: [email protected]

Running title: Inducing T-cell immunity overcomes resistance to checkpoint blockade

in pancreatic cancer Keywords: Pancreatic cancer, PD-1, CTLA-4, immunotherapy, CD40 Funding: NIH grants: R01 CA169123 (R.H.V. and B.Z.S.), T32 CA009140 (R.W.),

T32 HL007439 (K.T.B.), T32 HL007775 (D.L.B), P30 CA016520 (R.H.V.), U19 AI 082630, P01 AI112521 (E.J.W.) Stand Up To Cancer (R.H.V.) Pancreatic Action Cancer Network-AACR (R.H.V.) Translational Center of Excellence in Pancreatic Cancer of the Abramson Cancer Center (R.H.V., C.C., E.E.F.)

5999 words, 5 figures, 8 supplementary figures The authors declare no conflict of interest.

on April 17, 2018. © 2015 American Association for Cancer Research. cancerimmunolres.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 February 12, 2015; DOI: 10.1158/2326-6066.CIR-14-0215

http://cancerimmunolres.aacrjournals.org/

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Abstract:

Disabling the function of immune checkpoint molecules can unlock T-cell immunity against

cancer, yet despite remarkable clinical success with monoclonal antibodies (mAb) that block

PD-1 or CTLA-4, resistance remains common and essentially unexplained. To date, pancreatic

carcinoma is fully refractory to these antibodies. Here, using a genetically engineered mouse

model of pancreatic ductal adenocarcinoma in which spontaneous immunity is minimal, we

found that PD-L1 is prominent in the tumor microenvironment, a phenotype confirmed in

patients; however, tumor PD-L1 was found to be independent of IFNγ in this model. Tumor T

cells expressed PD-1 as prominently as T cells from chronically infected mice, but treatment

with PD-1 mAbs, with or without CTLA-4 mAbs, failed in well-established tumors, thus

recapitulating clinical results. Agonist CD40 mAbs with chemotherapy induced T-cell immunity

and reversed the complete resistance of pancreatic tumors to PD-1 and CTLA-4. The

combination of αCD40/chemotherapy plus PD-1 and/or CTLA-4 induced regression of

subcutaneous tumors, improved overall survival, and conferred curative protection from multiple

tumor rechallenges, consistent with immune memory not otherwise achievable. Combinatorial

treatment nearly doubled survival of mice with spontaneous pancreatic cancers although no

cures were observed. Our findings suggest that in pancreatic carcinoma, a non-immunogenic

tumor, baseline refractoriness to checkpoint inhibitors can be rescued by the priming of a T-cell

response with αCD40/chemotherapy.




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INTRODUCTION

Pancreatic ductal adenocarcinoma (PDA) is a lethal and aggressive disease with the lowest 5-

year patient survival rates of any tumor type routinely tracked (6%). The incidence of PDA is

rising, and it is projected to become the second leading cause of cancer death in the United

States by 2025 (1). PDA is distinguished by a dense desmoplastic stroma, rich in fibroblasts,

extracellular matrix, and inflammatory leukocytes (but few infiltrating effector T cells). Although

new combination chemotherapies are increasingly effective for PDA (2,3), tumor response rates

remain low and durability is short.

T cells are key mediators of antitumor immunity and regulate the outcome of tumor

immune surveillance (4). Critical to this regulation are lymphocyte inhibitory receptors such as

programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4

(CTLA-4), which restrain T-cell antitumor immunity (5-8). Monoclonal antibodies (mAb) that

block PD-1 or CTLA-4 induce T cell-dependent tumor regression in many experimental systems

(7,8). Unprecedented rates of tumor regressions have been observed in patients with melanoma

and multiple carcinomas following treatment with mAbs against CTLA-4, PD-1, or programmed

death-ligand 1 (PD-L1), a ligand for PD-1 (9-14). PD-1 engagement inhibits T-cell function

primarily during the T-cell effector phase (15-17). Its main ligands are PD-L1 and programmed

death-ligand 2 (18). PD-L1 is expressed on tumor cells as well as on tumor-associated

leukocytes, and antibody blockade of either PD-1 or PD-L1 can restore antitumor immunity in

murine cancer models (19-21). CTLA-4 is a crucial immune checkpoint regulator expressed on

T cells; loss of CTLA-4 leads to rampant lymphoproliferation, autoimmunity, and death in mice

(22). Expression of CTLA-4 is found on effector T cells but especially regulatory T cells (Treg);

CTLA-4 blockade in murine tumor models both inhibits negative signaling in effector cells and

depletes Tregs (23-25).

Mechanisms of PD-1 or CTLA-4 resistance are poorly understood. Pre-existing T-cell

antitumor immunity has been hypothesized as a prerequisite (26-29). The majority of cancer




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patients treated with these agents alone do not respond clinically, and some tumor types, such

as PDA, are fully refractory (12,30,31). Although combinations of αPD-1 and αCTLA-4 may

improve tumor response rates in melanoma, a large fraction of patients still fail to respond (32).

Tumor PD-L1 expression in some tumors correlates spatially with the presence of infiltrating

CD8+ T cells, suggesting that tumor cells upregulate PD-L1 in response to immune pressure, a

hypothesis termed adaptive immune resistance (33-35). CD8+ T cell-derived IFNγ may drive

PD-L1 expression in malignant cells (18,36). These data suggest that the efficacy of checkpoint

inhibitors may require the presence of an endogenous antitumor T-cell response. In fact, the

augmentation of antitumor T-cell responses with vaccines, peritumoral poly(I:C), or intratumoral

oncolytic virus has been shown to improve baseline responses to checkpoint inhibitors in murine

models (21,27,37,38).

In the studies reported here, we tested the hypothesis that failed immune recognition or

poor T-cell priming underlies weak clinical responses to checkpoint therapy in pancreatic

cancer, i.e. induction of T-cell immunity is required to potentiate tumor regressions not otherwise

achievable with checkpoint blockade alone. We studied the KPC mouse model of spontaneous

PDA in which expression of oncogenic KrasG12D and mutant p53 is targeted to the pancreas by

Cre recombinase under the control of the pancreas-specific promoter Pdx-1 (39). This model

recapitulates the molecular, histologic and immune parameters of the human disease (39-43).

Analysis of human PDA was performed to confirm the clinical relevance of our findings in the

murine model. We induced T-cell immunity using an agonistic αCD40 in combination with

chemotherapy (44,45), and studied the impact of αPD-1/αCTLA4 mAbs.




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MATERIALS AND METHODS

Mice

All animal protocols were reviewed and approved by the Institutional Animal Care and Use

Committee of the University of Pennsylvania. KrasLSL-G12D/+, Trp53LSL-R172H/+, Pdx1-Cre (KPC)

mice (39), and KrasLSL-G12D/+, Trp53LSL-R172H/+, Pdx1-Cre, LSL-Rosa-YFP (KPC-Y) mice (46) were

backcrossed 10 generations on the C57BL/6 background. Six- to eight-week-old female

C57BL/6 and B6.129S7-Ifngtm1Ts/J (IFNγ ko) mice used for implantable tumor studies were

from Jackson Laboratories.

Cell Lines

PDA cell lines from KPC or KPC-Y mice were derived from single-cell suspensions of PDA

tissue as previously described (42). Dissociated cells were plated in a 6-well dish with serum

free DMEM. After 2 weeks, media was changed to DMEM + 10% FCS. After 4-10 passages,

cells were used in experiments. The cell lines were tested and confirmed to be mycoplasma-

free. No other authentication assays were performed.

In vivo Mouse Studies

For implantable tumor experiments, PDA tumor cells (5x105) were injected subcutaneously in

PBS into the flanks of mice and allowed to grow 9-11 days until tumor volumes averaged 30-

100mm3. Mice were then enrolled into treatment groups such that cohorts were balanced for

baseline tumor size. Mice were treated intraperitoneally (i.p.) with αPD-1 (RMP1-14, BioXcell;

200μg per dose) on days 0, 3, 6, 9, 12, 15, 18, and 21 (after enrollment) and/or αCTLA-4 (9H10,

BioXcell; 200μg per dose) on days 0, 3, and 6. All antibodies were endotoxin free. Clinical grade

gemcitabine (Eli Lilly) was purchased through the Hospital of the University of Pennsylvania

Pharmacy; clinical grade nab-paclitaxel was either purchased or a kind gift from Celgene.

Chemotherapy vials were resuspended and diluted in sterile PBS, and injected i.p. at 120 mg/kg

(for each chemotherapeutic) on day 1. As a control for the human albumin component of nab-




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paclitaxel, control cohorts were treated with human albumin at the same dose as the albumin

component of nab-paclitaxel (108 mg/kg) on day 1 (Sigma Life Science). All antibodies were

given i.p. Agonistic αCD40 (FGK45, BioXcell; 100μg) was given on day 3. For T-cell depletion

studies, αCD8 (2.43, BioXcell; 200μg per dose) and αCD4 mAbs (GK1.5, BioXcell; 200μg per

dose) were injected twice weekly for the duration of the experiment, starting on day 0 (day of

enrollment). For isotype controls, rat IgG2a (2A3, BioXcell; 100μg) and rat IgG2b (LTF-2,

BioXcell; 200μg per dose) were used. This approach achieved >98% depletion of CD8+ and

CD4+ T cells in peripheral blood and tumor tissue compared to that of control mice, as

monitored by flow cytometry. For macrophage depletion studies, clodronate encapsulated

liposomes (CEL) or PBS encapsulated liposomes (PEL, both at 12μl/g; purchased from Dr. Nico

van Rooijen, Vrije Universiteit, Amsterdam, the Netherlands) were used i.p. starting on day -1

and repeated every 4 days for the duration of the experiment; in these experiments, 2.5x105

PDA cells were implanted. For tumor rechallenge studies, αCD8 or isotype control antibodies

were injected i.p. the day before the second rechallenge and continued twice weekly until day

60 or the mouse was sacrificed for tumor burden. To monitor growth of subcutaneous tumors,

tumor diameters were measured by calipers and volume calculated by 0.5 x L x W2 in which L is

the longest diameter and W is the perpendicular diameter. Endpoint criteria for the survival

studies included tumor volume exceeding 1,000 mm3 or tumor ulceration. Mice that died

suddenly or developed vestibular signs, as described in Supplementary Fig. S8, with minimal

tumor burden were censored on the day of death or euthanasia.

For studies using the KPC model, young KPC mice were monitored by abdominal

palpation and/or ultrasonography (Vevo 2100 Imaging System with 55MHz MicroScan

transducer, Visual Sonics) for the development of pancreatic tumors. Mice with ultrasound

diagnosed tumors of volume 30-150 mm3 were enrolled and block randomized into treatment

groups. Tumors were visualized and reconstructed for quantifying tumor volume using the

integrated Vevo Workstation software package. Baseline tumor volume was not significantly




7

different across cohorts. KPC mice were treated with the same dose and schedule of antibodies

and chemotherapeutics as noted above in the subcutaneous model. Mice were censored from

study if they developed a secondary malignancy (n=1). Endpoint criteria included tumor volume

exceeding 1,000 mm3 (by ultrasonography), severe cachexia, or extreme weakness and

inactivity.

For viral studies, C57BL/6 mice were infected intravenously with 4x106 PFU of LCMV

clone 13, which was propagated, titrated and used as previously described (15). Mice were

sacrificed on day 30 post infection and tissues harvested for analyses.

Collection of Tissue Samples from Mice

The entire pancreas (KPC) or subcutaneous tumor was washed in PBS, minced into small

fragments, and incubated in collagenase solution (1 mg/ml collagenase V in DMEM) at 37°C for

45 min. Dissociated cells were passed through a 70 μM cell strainer twice and washed three

times in DMEM. Spleens and lymph nodes were homogenized and passed through a 70 μM cell

strainer to achieve single cell suspensions. For spleens, red blood cells were lysed using ACK

Lysis Buffer (BioWhittaker).

Antibodies, Flow cytometry, In vitro IFN stimulation of tumor cells, and Toxicology are

described in Supplementary Methods.

Patient Samples and Analysis

Formalin-fixed, paraffin-embedded tissue samples were prepared after surgical resection of

patients with resectable pancreatic carcinoma according to an IRB-approved protocol, as noted

in Supplementary Methods.

Statistical Analysis




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Differences between two groups were analyzed by two-tailed Student’s T test. Differences

between three or more groups were analyzed by one-way ANOVA with Bonferonni’s multiple

comparison test used as a post hoc test to assess differences between any two groups. Tumor

growth curves were analyzed by two-way ANOVA, with Tukey multiple comparisons of means

used as a post hoc test to assess differences between any two groups. Survival curves were

assessed by Log-rank (Mantel-Cox). Correlation between two groups was assessed by

Spearman’s Rank Correlation Coefficient. All statistical analyses were performed on GraphPad

Prism 6 (GraphPad) except 2-way ANOVA and related post hoc testing which were performed

on R Statistical Software (R Core Team). p≤0.05 denotes differences that are statistically

significant.




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RESULTS

PD-1/PD-L1 axis is highly expressed in murine and human PDA

We interrogated the expression of PD-1 and PD-L1 using the KPC spontaneous genetic model

of PDA. Within the microenvironment of KPC tumors, few infiltrating T cells were observed, as

previously reported (41,42), but these T cells prominently expressed PD-1 in all subsets

including CD8+, CD4+, and regulatory (Foxp3+) T cells. For each subset, PD-1 expression was

significantly higher in the tumor than in the corresponding populations in the spleens of the

same tumor-bearing mice (Fig. 1A). In the absence of a distinct marker for pancreatic epithelial

cells in the KPC model, we identified tumor cells with negative gating, excluding leukocytes

(CD45), endothelial cells (CD31) and mesenchymal populations (CD90) by flow cytometry of

single-cell suspensions of KPC tumors. KPC pancreatic tumor cells exhibited moderate

expression of PD-L1 on more than 40% of the identified tumor cells (Fig. 1B). PD-L1 was also

expressed by 10%-50% of normal pancreatic epithelial cells identified in tumor-free wild-type

mice. Dendritic cells (DC) and macrophages in the KPC tumor microenvironment expressed

very high levels of PD-L1, statistically significantly higher compared to PD-L1 expression of

these same antigen-presenting cell (APC) populations in the spleens of KPC mice (Fig. 1B).

To assess whether these findings in the KPC model were consistent with human PDA,

we examined human PDA samples for PD-L1 expression. In primary tumors from patients with

resected PDA, we observed moderate to intense expression of PD-L1 on tumor and

mononuclear cells in 4 of 8 (50%) resection specimens (Fig. 1C and Supplementary Fig. S1).

We also observed that T cells in human PDA were relatively rare within malignant foci (mean

ratio of CD8+ T cells per μm2 of tumor vs. non-tumor areas was 0.065 + 0.052, range of 0.000-

0.170) – again consistent with the KPC model (42). PD-L1 expression on tumor cells in human

samples did not correspond spatially with the presence of CD8+ T cells; there was no statistical

correlation between intensity or extent of tumor PD-L1 expression and intratumoral CD8+ T-cell

infiltration (p=0.69) (Fig. 1C). For example, of the two tumors with the most intratumoral CD8+ T




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cells, one had intense and the other had minimal PD-L1 expression (Fig. 1C). These data in

human PDA are in contrast to the correspondence of tumor PD-L1 expression and T-cell

infiltration previously reported for tumors from patients with melanoma or kidney or head and

neck carcinoma (HNSCC) (33-35).

PD-1 is as highly expressed in murine PDA as it is in chronic LCMV infection

To evaluate the potential role of the PD-1/PD-L1 axis in mediating immune suppression in PDA,

we first generated a PDA cell line from a backcrossed KPC mouse and established

subcutaneous PDA tumors in immune competent C57BL/6 mice. Histopathologic examination of

established tumors from this model showed recapitulation of both the cellular and extracellular

components of spontaneous KPC tumors, with prominent deposition of a dense desmoplastic

stroma and comparable populations of infiltrating immunosuppressive leukocytes, including

F4/80+ macrophages (data not shown). We then examined expression of PD-1 on T cells from

subcutaneous tumor-bearing mice but did so by simultaneously examining PD-1 expression on

T cells from a parallel cohort of mice in which chronic lymphocytic choriomeningitis viral (LCMV)

infection had been established with LCMV clone 13 (Fig. 2A). In many ways, this model of

chronic LCMV infection has served as a gold standard for understanding the transcriptional

basis and phenotype of exhausted CD4+ and CD8+ T cells (15,16,47-50). Two of the most highly

upregulated genes mechanistically linked to T-cell exhaustion in response to chronic infection in

this model are PD-1 and Lag-3 (15). We therefore compared coexpression of these markers on

intratumoral and splenic T cells in mice bearing established subcutaneous PDA tumors with

splenic T cells from mice with chronic LCMV (Fig. 2A). Intratumoral CD8+, CD4+, and regulatory

T cells co-expressed PD-1 and Lag-3 at levels comparable to the same T-cell populations in

LCMV-infected mice (Fig. 2B). This phenotype was restricted to the tumor, as splenic T cells

from tumor-bearing mice did not co-express PD-1 or Lag-3. Thus, T-cell expression of PD-1 is

as prominent in the PDA tumor microenvironment as it is in chronic LCMV infection.




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In the subcutaneous PDA model, about 60% to 70% of tumor cells isolated from

established tumors expressed PD-L1 (Fig. 2C), similar to the expression of PD-L1 on this cell

line grown in vitro (Fig. 2D). These findings were confirmed using a YFP+ tumor cell line

established from a pancreatic tumor isolated from a KPC-Y genetically engineered mouse; in

this model, YFP serves as a validated lineage tracer for pancreatic epithelium (46). After

subcutaneous tumor implantation and growth in syngeneic hosts, we found that on average

66.7% of YFP+ tumors cells expressed PD-L1, as measured by flow cytometry (Supplementary

Fig. S2). Moreover, high levels of PD-L1 on both DCs and macrophages were observed in the

tumor microenvironment of the KPC subcutaneous tumors (Fig. 2C), mirroring PD-L1

expression on these APC subsets in spontaneous tumors of KPC mice. Both a higher

percentage of PD-L1+ APCs and a higher (~3-4-fold) mean fluorescence intensity (MFI) of PD-

L1 was observed compared to that of the corresponding APC populations in the spleens of the

same mice (Fig. 2C). These data indicate that PD-L1 expression is prevalent in the PDA

microenvironment.

Tumor PD-L1 expression in PDA is not IFNγ-dependent

PD-L1 expression in human melanoma and HNSCC correlates spatially with T-cell infiltration

(33,35), and in melanoma, tumor expression of PD-L1 is dynamically upregulated in response to

IFNγ secreted by infiltrating CD8+ T cells (36). To determine whether this same mechanism is

responsible for PD-L1 expression in PDA, we assessed the ability of our PDA cell line to

upregulate PD-L1 in response to IFNγ; in vitro, IFNγ stimulation resulted in increased

expression of PD-L1 by PDA cells (Fig. 2D). In vivo, we evaluated tumor and APC PD-L1

expression in the presence or absence of T cells and IFNγ. Subcutaneous PDA tumors were

established in mice that were genetically lacking IFNγ, systemically depleted of CD4+ and CD8+

T cells, or both. Tumor growth rate in vivo was the same for each condition compared to that of

control (data not shown). Analysis of these tumors showed no significant change in tumor PD-




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L1 expression (either percentage or MFI) with regard to IFNγ or T-cell status (Fig. 2E). Analysis

of the APC populations in these tumors indicated that IFNγ plays a minor role in the regulation

of PD-L1 expression on intratumoral DCs and macrophages. Small but statistically significant

differences were observed in the percentage and MFI of PD-L1 expression on intratumoral

APCs between IFNγ-sufficient and IFNγ-deficient hosts (Fig. 2F). In contrast, the presence or

absence of T cells did not affect PD-L1 expression by APCs regardless of host IFNγ status (Fig.

2F), recapitulating the lack of correspondence between CD8+ T cells and PD-L1 expression in

human PDA (Fig. 1C).

T-cell stimulation with CD40/gemcitabine/nab-paclitaxel converts PDA from being fully

refractory to highly sensitive to checkpoint blockade

We then tested the antitumor in vivo efficacy of PD-1-blocking mAbs either with or without

CTLA-4 blocking mAbs (Fig. 3A). CTLA-4 is expressed at higher levels on intratumoral CD8+

and CD4+ T-cell populations (including Treg) when compared to that of splenic populations,

suggesting that blockade of this negative checkpoint may improve antitumor immunity

(Supplementary Fig. S3). Even with αCTLA-4, αPD-1 did not impact tumor growth or survival

(Fig. 3B), even though a comparable αPD-1 dosing schedule reproducibly improves clinical

outcomes in mice chronically infected with LCMV clone 13 (50,51). This lack of antitumor

efficacy is similar to the lack of responses observed to date in patients with advanced PDA

treated with αPD-L1 or αCTLA-4 (12,30,31).

These same reagents have shown efficacy in patients with other malignancies; one

possible distinction may be the presence of an antitumor immune response at baseline in

subsets of these patients (28,52). We therefore hypothesized that the induction of a T-cell

response would be required to overcome refractoriness to αPD-1 and αCTLA-4 in PDA and

achieve clinical benefit. Agonist αCD40 antibody facilitates cancer vaccines (53) and can

synergize with chemotherapy-induced immunogenic cell death to initiate a T cell-dependent




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antitumor regression, providing a vaccine effect in model systems for which a tumor-rejection

antigen is not characterized (45,54). APCs in KPC-derived subcutaneous tumors express CD40

(Supplementary Fig. S4), as we have previously shown (42). Here, we studied gemcitabine and

nab-paclitaxel because this combination was recently approved by the FDA for the treatment of

metastatic PDA (3), and gemcitabine has been shown to cooperate immunologically with αCD40

(45). Treatment of mice with established subcutaneous PDA tumors with αCD40/chemotherapy

altered the phenotype of tumor-infiltrating T cells although the percent of T cells infiltrating the

tumors did not change. There were statistically significantly fewer CD8+ T cells that co-

expressed the inhibitory PD-1 and Lag-3 markers in treated tumors and more proliferating CD4+

and CD8+ T cells were found in the tumors of treated mice compared to that of controls

(Supplementary Fig. S5).

The combination of gemcitabine and nab-paclitaxel at the maximum tolerated dose did

not induce regression of established subcutaneous PDA tumors; however, the addition of

αCD40 to this chemotherapy regimen inhibited tumor growth and improved survival compared to

control-treated tumor-bearing mice (Fig. 3C). These effects were macrophage-independent as

CEL failed to abrogate the effect (Supplementary Fig. S6). Given that we have previously

observed that αCD40 plus chemotherapy in the subcutaneous KPC model manifests an

antitumor effect that is T-cell dependent (55), we added αPD-1, αCTLA-4, or both to treatment

with αCD40/chemotherapy in an effort to enhance T-cell immunity. The addition of αPD-1,

αCTLA-4, or both to αCD40/chemotherapy enhanced tumor growth inhibition, and led to an

increase in survival in mice bearing subcutaneous PDA tumors (Fig. 3C). Moreover, these

combinations led to complete rejection of established tumors and long-term tumor-free survival

in significant proportions of mice treated with the combined regimen (Fig. 3D). Treatment with

αPD-1 and αCTLA-4 did not alter CD40 expression on APCs in the tumor microenvironment

(Supplementary Fig. S4). The highest rates of tumor regression were observed in mice treated

with both αPD-1 and αCTLA-4, with 39% (17 of 44) of mice achieving long-term complete




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remission and survival after treatment with all three antibodies plus chemotherapy (Fig. 3D).

Tumor growth was delayed in nearly all mice treated with αCD40/chemotherapy and αPD-

1/αCTLA-4, even in those mice not completely rejecting their tumors, suggesting that the tumor

response rate is even higher than the tumor rejection rate in this model (Supplementary Fig.

S7). Although treatment was well tolerated in the vast majority of mice, in 6.3% of mice treated

with αCD40/chemotherapy and at least one checkpoint blocking mAb we noted clinical

deterioration consistent with an infectious syndrome (Supplementary Fig. S8).

Rejection of PDA tumors by αCD40/chemotherapy and checkpoint blockade is T cell-

mediated

To determine whether the antitumor effect we observed was T cell-mediated, we repeated the

study with a cohort of mice depleted of CD4+ and CD8+ T cells, starting on the day prior to the

initiation of therapy. In the absence of T cells, the treatment did not inhibit tumor growth or afford

survival advantage, and no T cell-depleted mice rejected the tumor or survived long-term (Fig.

4A).

To understand the effect of our treatment on intratumoral T-cell populations, we treated

cohorts of tumor-bearing mice with αPD-1/αCTLA-4, αCD40/chemotherapy, both, or neither

(control), and sacrificed mice one week after treatment with αCD40 (or control) to analyze

tumors for T-cell infiltration. Tumors from mice treated with αPD-1/αCTLA-4 plus

αCD40/chemotherapy had a significantly increased (7-fold) CD8:Treg ratio compared to that of

control-treated mice (Fig. 4B). This phenotype was also seen in some of the mice treated with

αCD40/chemotherapy or αPD-1/αCTLA-4, although neither one of these two groups exhibited

as consistent an increase in the CD8:Treg ratio as the mice treated with αPD-1/αCTLA-4 plus

αCD40/chemotherapy (Fig. 4B). The CTLA-4 mAb clone 9H10 partly mediates its antitumor

effect by depletion of Tregs, which express CTLA-4 (24); however, we observed that mice

treated with αPD-1/αCTLA-4 alone did not have a significantly decreased percentage of Tregs




15

among CD4+ T cells (Fig. 4B) or among total CD45+ cells. Rather, the administration of

αCD40/chemotherapy (either with or without αPD-1/αCTLA-4) was associated with a significant

decrease in Treg percentages compared to that of control treated mice; all groups treated with

immunotherapy demonstrated a slight increase in the CD8+ T-cell infiltrate, although the

changes were not statistically significant (Fig. 4B). These data suggest that

αCD40/chemotherapy changes the immune microenvironment in this PDA model and leads to a

decreased percentage of Tregs and increased CD8:Treg ratio, an effect that is augmented

further with the addition of checkpoint blockade. The greatest changes in Treg percentage and

CD8:Treg ratio were associated with the highest rates of complete remission and long-term

survival across cohorts reported in Fig. 3D.

To test whether mice that had completely rejected established PDA tumors had

developed immune memory, we rechallenged cohorts of mice that were in long-term complete

remission with the same number of cells of the same PDA tumor line but on the opposite flank

(Fig. 4C). We observed that 67% to 86% of such mice rejected the PDA tumor cells implanted

on the opposite flank without any additional therapy (Fig. 4C), consistent with those mice having

developed immunologic memory. Because the most likely effector memory T-cell population

mediating this effect is a CD8+ T cell, we further studied mice that had rejected both the initial

tumor and the first rechallenge on the opposite flank, and either depleted these mice of CD8+ T

cells or administered an isotype control antibody that did not deplete CD8+ T cells. All mice were

then rechallenged with the same number of cells of the same cell line on the original flank. All

mice depleted of CD8+ T cells rapidly developed progressively growing tumors at the site of the

second rechallenge, whereas 4 of 6 isotype-treated mice rejected this second tumor rechallenge

(Fig. 4D). This effect translated into a statistically significant difference in overall survival after

the second rechallenge (Fig. 4D).




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αCD40/chemotherapy cooperates with PD-1 blockade to improve survival of mice with

established tumors in the KPC genetic model of PDA

Having observed that the induction of T-cell immunity via αCD40/chemotherapy potentiates the

efficacy of checkpoint inhibitors in the subcutaneous model of PDA, we then tested this

approach in the autochthonous KPC model of PDA. Observations in the KPC model have

previously been shown to predict clinical responses in PDA patients treated with the same or

homologous agent (42,56-58). We therefore performed a randomized, controlled study of

checkpoint inhibition in combination with αCD40/chemotherapy in cohorts of tumor-bearing KPC

mice. Given the striking expression of PD-1 and PD-L1 in the KPC tumor microenvironment, we

chose to test our hypothesis using the αPD-1 mAb. Mice diagnosed with pancreatic tumors of

30 mm3-150 mm3 were randomized to treatment with αCD40 plus gemcitabine/nab-paclitaxel,

αPD-1, αCD40/chemotheray plus αPD-1, or control (as described in Materials and Methods and

summarized in Fig. 5A), using the same dose and schedule as used for mice in the

subcutaneous PDA studies. We observed a statistically significant increase in overall survival

for mice receiving αCD40/chemotherapy plus αPD-1 compared to that of control (p=0.015, log-

rank Mantel-Cox) (Fig. 5B). The effect was large: combination treatment nearly doubled the

median overall survival from 23 days in the control arm to 41.5 days in the experimental arm

with a hazard ratio of 0.334 (0.0584-0.657, 95% confidence interval). Neither PD-1 alone nor

αCD40/chemotherapy significantly improved overall survival. These data suggest that as

predicted by our findings in the subcutaneous PDA model, the induction of a T-cell response is

needed for the αPD-1 antitumor effects in PDA.




17

DISCUSSION

The clinical success of checkpoint inhibitors, including FDA approval of ipilimumab and

pembrolizumab for melanoma, has prompted investigations to replicate these results more

broadly in oncology. Early findings, however, suggest that many tumors are resistant, with

pancreatic carcinoma appearing completely refractory to monotherapy with checkpoint blockade

(12,30,31). Here, using a genetically engineered mouse model of PDA, which like human PDA

exhibits minimal spontaneous immunity, we demonstrate that despite robust expression of PD-1

and PD-L1 in the tumor microenvironment, treatment with αPD-1 with or without αCTLA-4 has

minimal antitumor impact, replicating that in PDA patients treated with analogous agents

(12,30,31). However, in the context of αCD40/chemotherapy, we demonstrate that the induction

of T-cell immunity converts PDA from a tumor that is completely refractory to αPD-1 and/or

αCTLA-4 into one in which checkpoint blockade controls tumor growth and significantly

improves survival in a CD8+ T cell-dependent manner. In particular, the combined regimen of

αCD40/chemotherapy plus αPD-1 nearly doubles the median overall survival in genetically

engineered KPC mice with pre-established spontaneous pancreatic tumors. Moreover, the

capability of the treated mice to reject second and third subcutaneous tumor challenges in a

CD8+ T cell-dependent fashion thereby rendering long-term survival, suggests that the

combined regimen induced the establishment of antitumor immune memory with curative

potential. These findings indicate that poorly immunogenic tumors, epitomized by the KPC

pancreatic tumor model, can nevertheless be controlled by the adaptive immune system

provided a dual approach of therapeutic T-cell induction and checkpoint blockade is utilized.

Immunologically, the PDA tumor microenvironment is considered especially suppressive,

but increasingly, there is an appreciation from studies in KPC and other PDA models of an

underlying sensitivity of PDA tumor cells to T-cell cytotoxicity (59). Unlike melanoma, PDA does

not commonly present with a robust tumor infiltration of CD8+ T cells (60-63). In genetically

engineered mouse models of PDA, a prominent network of immunosuppression is dominant




18

even at the earliest stages of disease (40-43,64). We demonstrated that PD-1 is as prominent in

the KPC tumor microenvironment as it is systemically in mice chronically infected with LCMV

(50). We propose therefore, that the lack of responses to treatment with checkpoint inhibitors in

KPC mice likely reflects a tumor microenvironment without an underlying antitumor T-cell

response.

The lack of PDA immunogenicity does not necessarily indicate an inherent lack of

antigenicity of PDA tumors cells; indeed, PDA cells might be unexpectedly sensitive to T-cell

killing because they have not been exposed to Darwinian-like T-cell selective pressure in vivo.

Without T-cell pressure, T-cell escape and classical immunoediting may not be necessary for

pancreatic tumor growth as it is for highly immunogenic tumors (4,59). Thus, in this study we

interpret the antitumor effects of αCD40/chemotherapy plus αPD-1/αCTLA-4 as a strategy that

overcomes acquired immune privilege in PDA. Other pathways and cells in the PDA tumor

microenvironment may also be “targetable” as part of novel immunotherapeutic approaches

(43,65,66).

Despite the 80% increased overall survival observed in KPC mice treated with

αCD40/chemotherapy and αPD-1 compared to that of the controls, all mice succumbed to their

disease. It is worth noting that there are no published reports of cures of KPC mice bearing

established invasive tumors. A few groups have reported improved overall survival (without

cures) with treatment in this model, including the recent demonstration that the vitamin D

analogue calcipotriol improves survival by 57% (57,67,68). Given the difference we observed in

the response to treatment between the subcutaneous and KPC PDA models, we hypothesize

that there is additional complexity in the tumor microenvironment of spontaneous KPC tumors

which limits therapeutic responses. Potential other immunosuppressive pathways contributing to

treatment resistance include myeloid-derived suppressor cells (MDSC), macrophages, and

fibroblast-activating protein+ (FAP+) mesenchymal cells, among others (43,65,66,69).




19

Our observations that αCD40/chemotherapy converts PDA from a tumor fully refractory

to checkpoint inhibition to one that is sensitive are important in the context of prior efforts to

extend the therapeutic range of αPD-1 and αCTLA-4. Our goal was to use a murine model that

reproduces the lack of clinical effect observed to date with αPD-1 and αCTLA-4 in PDA, in

contrast to many tumor models that exhibit baseline levels of responsiveness to αCTLA-4, αPD-

1, αPD-L1 or combinations of these agents. For example, in models of colon carcinoma,

melanoma, ovarian cancer, bladder cancer, and neuroblastoma, checkpoint therapy alone

inhibits tumor growth, improves survival, and occasionally mediates complete rejection (21,70-

73). In studies using the Panc02 subcutaneous PDA model, tumor rejection was observed in

50% of mice after treatment with αCTLA-4 (74), and treatment with αPD-L1 (with or without

gemcitabine) leads to significant decreases in tumor growth (75), findings not representative of

the clinical record of these agents in patients with PDA (12,30,31) and possibly related to

Panc02 being a carcinogen-induced and likely hypermutated tumor [whereas human PDA is not

a hypermutated tumor (76)]. Previous work in these types of models demonstrates that T-cell

stimulatory therapies can improve baseline effects of checkpoint blockade (21,27,37,38). In

PDA, the stimulation of a T-cell response using αCD40/chemotherapy is able to transform a

tumor that is refractory to checkpoint inhibition into a sensitive one, rather than only improving

upon baseline activity of checkpoint mAbs.

In the absence of a defined tumor antigen in our KPC model, we therapeutically induced

T cells with chemotherapy followed by an agonist αCD40 (45). Although other agents may also

synergize with αCD40 (77), gemcitabine in particular cooperates with αCD40 (45). Here, we

added nab-paclitaxel given the recent regulatory clinical approval of the combination. We have

previously demonstrated in KPC mice with spontaneous tumors that αCD40 can reprogram

macrophages to mediate (T cell-independent) stromal involution and short-term decreases in

tumor volume without improvement in overall survival (42); here, we found that a one-time

treatment with chemotherapy followed by αCD40 enabled a macrophage-independent T-cell




20

response, engendering immune memory and durable complete remissions in a large fraction of

mice harboring subcutaneous PDA tumors – and extending survival in KPC mice with

spontaneous tumors. Both the use of nab-paclitaxel and the subcutaneous setting may be

responsible for the differences in these observations, the subject of current investigations.

Although of interest, we could not evaluate in Fc receptor-null mice whether Treg changes from

αPD-1/αCTLA-4 were Fc receptor-dependent because the effects of agonistic αCD40 are lost in

such mice (78).

In certain human cancers, tumor cell PD-L1 expression co-localizes with lymphocytic

immune infiltrates, suggesting adaptive resistance by the tumor and the potential root of

sensitivity to αPD-1 (33-35,79). In the case of melanoma, tumor cell PD-L1 expression has been

demonstrated to be dependent on CD8+ T cells and the secretion of IFNγ (36). In contrast, we

found in our PDA mouse models harboring minimal intratumoral T cells, tumor cells express PD-

L1 at moderate levels and tumor-associated DCs and macrophages express high levels of this

inhibitory ligand. We further observed that PD-L1 expression in murine PDA is not dependent on

T cells or IFNγ, indicating that PD-L1 tumor expression does not appear to be an adaptive

response to immune pressure. These findings in the KPC model are corroborated by our

observations in human PDA in which there was no spatial correlation between tumor PD-L1

expression and the presence of intratumoral CD8+ T cells. Tumor PD-L1 expression has been

reported to be regulated by oncogenes such as EGFR (80), suggesting that the regulation of

PD-L1 in PDA may differ from the regulation of PD-L1 in other solid malignancies.

In summary, induction of T-cell immunity reduces resistance to PD-1 and CTLA-4

blockade and improves survival in pancreatic carcinoma. It is notable that four out of the five

reagents that we used in these studies have an FDA-approved human homologue (gemcitabine,

nab-paclitaxel, αPD-1, αCTLA-4) which should facilitate clinical translation.




21

ACKNOWLEDGEMENTS

RW designed study concept, designed and performed experiments, analyzed data, and wrote

and edited the manuscript. KTB, RAE, PMO, ARLM, DLB, and CC designed and performed

experiments, analyzed data, and edited the manuscript. BZS, EEF and EJW designed

experiments, analyzed data, and edited the manuscript. RHV designed study concept, designed

experiments, analyzed data, wrote and edited the manuscript, and supervised the study. We

thank Amy Ziober, Michael Feldman, Erin Dekleva, Tim Chao, Andrew Rech, Mark Diamond,

Steve Albelda, Jim Riley, Ellen Pure, and Anil Rustgi (Penn) and Dafna Bar-Sagi (New York

University) for helpful discussions.




22

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http://www.ncbi.nlm.nih.gov/pubmed/?term=Quigley%20M%5BAuthor%5D&cauthor=true&cauthor_uid=24777248

http://www.ncbi.nlm.nih.gov/pubmed/?term=Henning%20KA%5BAuthor%5D&cauthor=true&cauthor_uid=24777248

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FIGURE LEGENDS

Figure 1. Expression of PD-1 and PD-L1 in murine and human PDA

(A) Representative histograms and quantification of PD-1 expression on tumor-infiltrating CD8+

(gated on live, CD45+, CD3+, CD8+), CD4+ (gated on live, CD45+, CD3+, CD4+), or regulatory

(Tregs; gated on live, CD45+, CD3+, CD4+, FoxP3+) T cells in tumors (n=6-11) or spleens (n=4-

17) from tumor-bearing KPC mice. **p≤ 0.01, ****p≤ 0.0001.

(B) Representative histograms and quantification of PD-L1 expression on tumor cells, normal

pancreatic epithelial cells (gated on live, CD45neg, CD31neg, CD90neg), dendritic cells (DCs; gated

on live, CD45+, F4/80neg, CD19neg, CD11c+), and macrophages (Macs; gated on live, CD45+,

F4/80+) in tumors (n=4-11) or spleens (n=25) from tumor-bearing KPC mice; and normal

pancreata (n=5) from healthy C57BL/6 mice. **p≤ 0.01, ***p≤ 0.001.

(C) Histology and quantification of PD-L1 expression and CD8+ T-cell infiltration in human

pancreatic cancer sections. Left two panels, PD-L1 expression on malignant cells of a PDA

tumor (PD-L1 expression score of 4+ (intense), see Materials and Methods and Supplementary

Fig. S1; 40x and 400x magnification for top and bottom panels, respectively). Right top panel,

CD8 expression in serial section of the tumor in left panel, demonstrating few tumor-infiltrating

CD8+ T cells (40x magnification). Right bottom panel, plot describing correlation between

intratumoral CD8 count and tumor PD-L1 score (n=8). p=0.69.




28

Figure 2. PD-1/PD-L1 axis is highly expressed and is not IFNγ-dependent in a

subcutaneous murine model of PDA

(A) Experimental design for establishment of subcutaneous PDA tumors or chronic LCMV clone

13 (Cl-13) infection simultaneously in 2 cohorts of C57BL/6 mice.

(B) Representative flow plots and quantification of co-expression of PD-1 and Lag-3 on CD8+

(gated on live, lymphocytes, B220neg, NK1.1neg, CD8+), CD4+ (gated on live, lymphocytes,

B220neg, NK1.1neg, CD4+) and regulatory (Tregs; gated on live, lymphocytes, B220neg, NK1.1neg,

CD4+, FoxP3+) T cells from spleens of mice infected with LCMV Cl-13 (day 30) or the tumors

and spleens of mice bearing PDA tumors (day 14).

(C) Representative histograms and quantification of PD-L1 expression on tumor cells, dendritic

cells (DC), and macrophages (Mac) in subcutaneous PDA tumors or spleens from the same

mice (day 14), gated as in Figure 1B. (MFI=mean fluorescence intensity). ****p≤ 0.0001. See

also Supplementary Fig. S2.

(D) Histogram of KPC-derived PDA cell line interrogated for PD-L1 expression in vitro with or

without IFNγ in the culture, representative of 3 experiments.

(E) Quantification and MFI of PD-L1 expression on tumor cells from subcutaneous PDA tumors

established in either C57BL/6 (B6) or IFNγ-/- (IFNγ ko) mice with or without CD4+ and CD8+ T-

cell depletion (TCD) (day 16; n=6-8 mice per cohort).

(F) Quantification and MFI of PD-L1 expression on dendritic cells and macrophages in

subcutaneous PDA tumors grown in either B6 or IFN-γ ko mice with or without TCD (day 16;

n=6-8 mice per cohort). One-way ANOVA: %DCs PD-L1+, p=0.015; DC PD-L1 MFI, p=0.0039;

%Macs PD-L1+, p=0.58; Macs PD-L1 MFI, p=0.0007. Post hoc test p values are indicated

where statistically significant as *p≤ 0.05, **p≤ 0.01.




29

Figure 3. T-cell stimulation with αCD40/gemcitabine/nab-paclitaxel potentiates efficacy of

checkpoint blockade in murine model of PDA

(A) Experimental design for experiments of subcutaneous PDA tumors treated with checkpoint

inhibitors and αCD40/chemotherapy, as further described in Materials and Methods.

(G=gemcitabine; nP=nab-paclitaxel; q3d= antibody administered every 3 days).

(B) Tumor growth and survival analyses of mice bearing subcutaneous PDA tumors treated as

indicated (n=9-10 per cohort; results for control and αPD-1+αCTLA-4 cohorts representative of

3 independent experiments). See also Supplementary Fig. S7.

(C) Tumor growth and survival analyses of mice bearing subcutaneous PDA tumors treated as

indicated (n=9-10 per cohort; findings representative of 3 independent experiments). Two-way

ANOVA: p≤0.0001. Post hoc test p values indicated where statistically significant as *p≤ 0.05,

***p≤ 0.001, ****p≤ 0.0001. See also Supplementary Fig. S7.

(D) Percentage of mice bearing subcutaneous PDA tumors treated with indicated regimens that

rejected their tumors and survived tumor-free long-term (median follow-up of 42 days, range 23

to 222 days). Data compiled from 5 independent experiments.




30

Figure 4. Rejection of PDA tumors after αCD40/chemotherapy and checkpoint blockade

is T cell-mediated

(A) Tumor growth and survival analyses of mice bearing subcutaneous PDA tumors treated as

indicated (n=9-10 per cohort; G=gemcitabine; nP=nab-paclitaxel; TCD=CD4/CD8 depletion).

(B) Flow cytometric analysis of subcutaneous PDA tumors treated as indicated (day 18 after

tumor injection, day 7 after αCD40 treatment; P=αPD-1; C=αCTLA-4). One way ANOVA %CD8s

of live cells: p=0.17; one-way ANOVA %Tregs of CD4+ T cells: p=0.0004; one-way ANOVA

CD8:Treg Ratio: p=0.0005. Post hoc test p values indicated where statistically significant as *p≤

0.05, **p≤ 0.01, ***p≤ 0.001.

(C) Experimental design for 1st tumor rechallenge experiments. Table quantifies fraction and

percentage of mice that rejected tumor rechallenge in mice that had rejected the initial tumor

implantation and were tumor-free for at least 43 days. Data compiled from 3 independent

experiments.

(D) Experimental design for 2nd tumor rechallenge experiment. The 2nd rechallenge occurred on

day 31-49 after the first rechallenge. Table quantifies fraction and percentage of mice that

rejected the 2nd tumor rechallenge in mice that had rejected a 1st tumor rechallenge. Host mice

in this experiment were either treated with αCD8 (n=11) or isotype (Iso; n=6) antibodies.

Survival analysis of mice after the 2nd rechallenge with or without CD8 depletion is shown. Data

compiled from 2 independent experiments.




31

Figure 5. Combination of αCD40/chemotherapy and PD-1 blockade improves survival in

KPC genetic model of PDA

(A) Experimental design for randomized, controlled study in tumor-bearing KPC mice, treated

with αCD40/chemotherapy and αPD-1, as described in Materials and Methods. G=gemcitabine;

nP=nab-paclitaxel; q3d= antibody administered every 3 days.

(B) Overall survival analysis of tumor-bearing KPC mice treated as indicated (n=6-8 per cohort).

αPD-1 alone vs. isotype alone p=0.39; CD40/G/nP vs. isotype alone p=0.76; CD40/G/nP + αPD-

1 vs. isotype alone p=0.015.

(C) Median overall survival of tumor-bearing KPC mice as indicated.




Tumor CD8s

Isotype

PD-1

Spleen CD8s

A

B

C

Tumor CD4s Spleen CD4s

Tumor DCs Tumor Macs Tumor cells

Isotype

PD-L1

Pancreas Spleen DCs Spleen Macs

***

Tumor DCs Spleen DCs Tumor Macs Spleen Macs

**

Tumor Spleen

****

Tumor Spleen

****

Tumor Pancreas

Tumor Tregs Spleen Tregs

Winograd et al., Figure 1

PD-L1 CD8

Tumor Spleen

**




A

B

C

D

Isotype

PD-L1

Tumor cells Tumor Macs Tumor DCs Spleen DCs Spleen Macs

****

Tumor DCs Spleen DCs

****

Tumor DCs Spleen DCs

****

Tumor Macs Spleen Macs

****

Tumor Macs Spleen Macs

PD

-1

Lag-3

Tumor Tumor Spleen Cl-13 Spleen

E p=0.078

B6 B6

TCD

IFN-γ ko

TCD

IFN-γ ko

F


Day: 0 16

LCMV infection

Tumor injection

30

Sacrifice C57BL/6

Sacrifice

C57BL/6

Isotype

PD-L1

PD-L1 (IFN-γ)

Tumor cell line

p=0.076

B6 B6

TCD

IFN-γ ko

TCD

IFN-γ ko

*

B6 B6

TCD

IFN-γ ko

TCD

IFN-γ ko

*

** **

*

B6 B6

TCD

IFN-γ ko

TCD

IFN-γ ko

p=0.58

B6 B6

TCD

IFN-γ ko

TCD

IFN-γ ko

* **

B6 B6

TCD

IFN-γ ko

TCD

IFN-γ ko


Cl-13 Spleen

17% 16%

65% 2%

Tumor

23% 18%

3% 56%

Cl-13 Spleen

23% 23%

3% 51%

Tumor

31% 2%

22% 45%

Cl-13 Spleen

25%

52%

19%

4%

Tumor

50%

22%

25%

3%

Tumor cells

CD8+ T cells CD4+ T cells Regulatory T cells

Winograd et al., Figure 2 on April 17, 2018. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from



p=0.19

A

Treatment Cohort Long term survivors (%)

Isotype Alone 0/25 (0%)

PD-1 Alone 0/16 (0%)

CTLA-4 Alone 0/10 (0%)

PD-1+CTLA-4 1/19 (5%)

CD40/G/nP 3/25 (12%)

CD40/G/nP+PD-1 6/23 (26%)

CD40/G/nP+CTLA-4 7/22 (32%)

CD40/G/nP+PD-1+CTLA-4 17/44 (39%)

B

p=0.061

p<0.0001

D

0

Tumor injection

10 11 Day:

Mice enrolled

αPD-1, αCTLA-4 Treatment:

αCD40

13

G, nP

αPD-1, αCTLA-4

16

//

αPD-1, αCTLA-4

19

αPD-1 q3d

G:gemcitabine; nP:nab-paclitaxel

C57BL/6

***

****

**** **** ****

*

p<0.0001

C

Winograd et al., Figure 3 on April 17, 2018. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from



Treatment

Cohort

Reject 2nd

rechallenge

αCD8 Iso

PD-1+CTLA-4 ND ND

CD40/G/nP ND 1/2

CD40/G/nP+P 0/3 1/2

CD40/G/nP+C 0/3 2/2

CD40/G/nP+P+C 0/5 ND

A

B

D

C

p<0.0001

0

Tumor injection

10 Days:

Mice enrolled

52-124 // //

1st tumor rechallenge

31

End of treatment

Treatment

Cohort

Reject 1st

rechallenge

PD-1+CTLA-4 0/1 (0%)

CD40/G/nP 2/3 (67%)

CD40/G/nP+P 5/6 (83%)

CD40/G/nP+C 6/7 (86%)

CD40/G/nP+P+C 6/9 (67%)

G:gemcitabine; nP:nab-paclitaxel;

P:αPD-1; C:αCTLA-4

p<0.0001

C57BL/6

***

* *


1

αCD8 or Iso twice weekly

2nd tumor rechallenge

C57BL/6

Days: 0

Mice that

rejected 1st

rechallenge

p=0.0024

CD40/G/nP

+P+C

P+C CD40/G/nP Control CD40/G/nP

+P+C

P+C CD40/G/nP Control

** **

CD40/G/nP

+P+C

P+C CD40/G/nP Control




A

B

0 1 Day:

Mice enrolled

αPD-1

Treatment: αCD40

3

G, nP

αPD-1

6

αPD-1

9

αPD-1 q3d Tumor-bearing

KPC

C Median Overall Survival

Isotype Alone PD-1 Alone CD40/G/nP CD40/G/nP + PD-1

23 29 30.5 41.5


G:gemcitabine; nP:nab-paclitaxel




Published OnlineFirst February 12, 2015.Cancer Immunol Res Rafael Winograd, Katelyn T Byrne, Rebecca A Evans, et al. carcinomaPD-1 and CTLA-4 blockade and improves survival in pancreatic Induction of T cell immunity overcomes complete resistance to

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