María E. Rodriguez-Ruiz 1,2 1 - Cancer Researchcancerres.aacrjournals.org/content/canres/early/2016/08/20/0008... · 1 Abscopal effects of radiotherapy are enhanced by combined immunostimulatory

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Abscopal effects of radiotherapy are enhanced by combined immunostimulatory mAbs and are dependent on CD8 T cells and crosspriming.

María E. Rodriguez-Ruiz 1,2, Inmaculada Rodriguez 1, Saray Garasa 1, Benigno Barbes 1, Jose Luis Solorzano 2, Jose Luis Perez-Gracia 2, Sara Labiano 1, Miguel F.

Sanmamed1,3, Arantza Azpilikueta 1, Elixabet Bolaños 1, Alfonso R. Sanchez-Paulete 1,

M. Angela Aznar 1, Ana Rouzaut 1, Kurt A. Schalper4, Maria Jure-Kunkel5, Ignacio

Melero 1,2.

1Division of Immunology and Immunotherapy, Center for Applied Medical Research

(CIMA), University of Navarra and Instituto de Investigacion Sanitaria de Navarra

(IdISNA).Pamplona, Spain.

2University Clinic, University of Navarra and Instituto de Investigacion Sanitaria de

Navarra (IdISNA). Pamplona, Spain.

3Department of immunobiology, Yale School of Medicine, New Haven, CT.

4Departments of Pathology and Medicine (Medical Oncology), Yale School of

Medicine, New Haven, CT

5Bristol-Myers Squibb. Lawrenceville, NJ.

Running title: Radioimmunotherapy and abscopal effects.

Key words: Abscopal effects, PD-1, CD137, radiotherapy

Financial support: This work was financially supported by grants from MICINN (SAF2011-22831 and SAF2014-52361-R). I. Melero was also funded by the Departamento de Salud del Gobierno de Navarra, Redes temáticas de investigación cooperativa RETICC, European Commission VII Framework and Horizon 2020 programs (AICR and PROCROP), SUDOE-IMMUNONET, Fundación de la Asociación Española Contra el Cáncer (AECC), Fundación BBVA and Fundación Caja Navarra. ME Rodriguez-Ruiz receives a Rio Hortega contract from ISCIII. S. Labiano is recipient of predoctoral scholarship from MICINN.

Conflict interests: M J-K is a full time employee in Bristol Myers. IM has served as a consultant for Bristol-Myers, Roche-Genentech, AstraZeneca, Incyte, Alligator and receives research grants from Pfizer and Bristol Myers Squibb.

Research. on September 17, 2018. © 2016 American Association for Cancercancerres.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 August 22, 2016; DOI: 10.1158/0008-5472.CAN-16-0549

http://cancerres.aacrjournals.org/

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Corresponding authors: Ignacio Melero and Maria E. Rodríguez-Ruiz. [email protected] and [email protected]. CIMA and CUN. Universidad de Navarra. Av.Pio XII,55.31008.Pamplona.

Word counts

Total Abstract words: 237

Total word count 5,327

Total Figures: 7

Total References: 52




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Abstract

Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can

be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to

act both on irradiated tumor lesions and on distant, non-irradiated tumor sites. The

combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs

was conducive to favorable effects on distant non-irradiated tumor lesions as observed

in transplanted MC38 (colorectal cancer), B16OVA (melanoma) and 4T1 (breast

cancer) models. The therapeutic activity was crucially performed by CD8 T cells, as

found in selective depletion experiments. Moreover, the integrities of BATF-3-

dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to

CD8+ T cells and of the type I interferon system were absolute requirements for the

antitumor effects to occur. The irradiation regimen induced immune infiltrate changes

in the irradiated and non-irradiated lesions featured by reductions in the total content of

effector T cells, Tregs, and myeloid-derived suppressor cells (MDSC), while effector T

cells expressed more intracellular IFNγ in both the irradiated and contralateral tumors.

Importantly, 48h following irradiation CD8+ TILs showed brighter expression of

CD137 and PD1, thereby displaying more target molecules for the corresponding

mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lymphocytes from

surgically excised human carcinomas that were irradiated ex-vivo. These mechanisms

involving crosspriming and CD8 T cells advocate clinical development of

immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be

synergistically accompanied by radiotherapy strategies, even if disease is left outside

the field of irradiation.




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Introduction

Radiotherapy is a solid pillar of cancer treatment used to treat localized stages of

a broad variety of malignant diseases and to alleviate local complications in advanced or

metastatic cases as a palliative treatment. The mechanism of action of ionizing

radiotherapy against cancer is thought to mainly rely on catastrophic damage of

genomic DNA, leading to apoptotic tumor cell death. Many cellular genetic and

epigenetic factors affect the sensitivity of each tumor to radiotherapy approaches.

Recently, the tumor stroma component has been found to play a key role in the outcome

of irradiated tumors (1). When radiotherapy is prescribed to a patient, it is assumed that

normal non-malignant tissue will also be irradiated giving rise to multifarious biological

effects including inflammation and scarring (1). Radiotherapy can be performed by

applying an external beam of irradiation or by the temporal surgical insertion of

radiation sources guided by catheters into the cancer tissue using techniques collectively

known as brachytherapy.

Immunotherapy is emerging as another major pillar for the treatment of cancer

treatment. Monoclonal antibodies acting on immune system receptors to derepress or

agonistically augment antitumor immunity are being developed in the clinic (2).

Antibodies against the inhibitory (checkpoint) receptor CTLA-4 were the first to be

clinically developed with ipilimumab receiving FDA and EMA approval for metastatic

melanoma (3). Among these checkpoint inhibitor monoclonal immunostimulatory

antibodies, agents blocking the PD1/PD-L1 receptor/ligand pair have already attained

FDA and EMA approval for metastatic melanoma(4), NSCLC (5-7), renal cell

carcinoma (8) and other indications are under regulatory evaluation. This achievement

was preceded by extensive and successful preclinical research in mouse models.




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Agonist antibodies crosslinking CD137 (4-1BB) were also shown to enhance

antitumor immunity in mice to the point of causing the rejection of transplanted tumors

(9). Two antibodies against CD137 are undergoing phase II clinical trials with

promising results (10, 11). Anti-PD1 and anti-CD137 mAb act on T cells that express

these receptors on their plasma membrane presumably as a consequence of an antigen-

cognate activation process. Hence, the main mechanism of action is exerted on tumor

infiltrating lymphocytes that express such receptors on their surface, thus becoming

amenable to pharmacological modulation with the corresponding mAb. In preclinical

mouse models, anti-CD137 and anti-PD1 mAbs exert powerful synergistic effects (12),

that have given rise to two ongoing clinical trials testing such a combination

(NCT02253992, NCT02179918).

The interphase between radiotherapy and immunotherapy is an exciting

emerging topic. Radiotherapy causes biological effects known to both ignite (13, 14)

and quench the cellular immune response (13, 14). The type of cell death induced by

radiotherapy is considered immunogenic (15, 16), because it sets in motion multiple

alarmins (15, 16) and proinflammatory mechanisms (17). Radiotherapy-induced cell

death is a potential source of tumor antigens to be uptaken, processed and presented by

dendritic cells to CD8+ T lymphocytes, a processs that is collectively known as

crosspresentation (22 (18) and termed crosspriming if it results in CTL activation.

Crosspresentation to CD8+ T cells is mainly mediated by a specialized subset of

dendritic cells which are dependent for development on the Batf-3 transcription factor

(19) and on sFLT-3L as a growth factor. We have published that this DC subset is

critical for the therapeutic effects of anti-PD1 and anti-CD137 mAbs by means of

crosspresentation of tumor antigens (20). This DC subset is also known to be involved

in eliciting post-radiotherapy CTL immune responses (21). However, other mechanisms




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such as irradiation-dependent TGFβ production and myeloid cell recruitment are

considered immunosuppressive.

Immunostimulatory monoclonal antibodies have already been combined with

radiotherapy in preclinical models. Anti-CTLA4 mAb (22), anti-PD1 mAb (23, 24) and

anti-CD137 mAb (25-27), show evidence for synergistic effects with external beam

irradiation. Furthermore, triple combinations of radiotherapy with anti CTLA-4 plus

anti PD1 exert efficacious synergistic effects against B16F10 melanoma tumors as seen

against the directly irradiated tumor and a concomitant tumor, implanted outside the

irradiation field (28), a phenomenon known as the abscopal effect of radiotherapy (29).

Anecdotal evidence in the clinic suggests that in a patient treated with anti

CTLA-4 mAb (ipilimumab) and subsequent palliative radiotherapy there were objective

responses outside the irradiation field, concurrent with increases in the titer of

antibodies against the shared tumor antigen NY-ESO1 (30). In a phase II clinical trial

testing the ipilimumab plus radiotherapy combination there was a trend towards better

overall survival in metastatic melanoma patients (28).

In this study, we use different mouse models to demonstrate that external beam

radiotherapy synergizes with immunostimulatory anti-PD1 and anti-CD137 mAbs as

single agents and when used in combination. The therapeutic effects were attributed to

CD8 T cells by depletion experiments and involved profound changes in the tumor

microenvironment that include an augment in the expression of the receptors to be

targeted by the immunomodulatory mAb.




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Materials and Methods

Cell lines.

Tumor cells lines, MC38, a colon adenocarcinoma cell line of C57BL/6 origin

whose identity (Columbia, MO, USA. Case 6592-2012) was provided to us by Dr. Karl

E. Hellström (Seattle, WA). 4T1 breast carcinoma cells of BALB/c origin were a kind

gift from Dr. Sandra Demaria who sent an authenticated vial from the master cell bank

at NYU (New York City). B16F10-OVA melanoma-derived cells, that are transfected to

express chicken ovalbumin (OVA) have been verified by Idexx Radil in 2012 and kept

as a master cell bank vials thawn every 3-6 months and were cultured in RPMI 1640

supplemented with 10% fetal bovine serum (FBS), 2mmol/Ll-glutamine, 0.05 mmol/L 2

mercaptoethanol, HEPES, penicillin, and streptomycin at 37º in a humidified

atmosphere containing 5% CO2. All these cells lines were certified as being free of

contamination by Mycoplasma using the Mycoplasma detection kit (MycoAlert

Mycoplasma Detection Kit from Lonza).

In vivo tumor experiments

C57BL/6 female mice were injected s.c. with 5×105 MC38 and 5×105 B16OVA cells,

respectively, in the right flank (primary tumor) and with 3×105 MC38 and 3×105

B16OVA cells in the left flank (secondary tumor). A similar scheme was used to

subcutaneously engraft 4T1 cells in female BALB/c mice. Perpendicular tumor

diameters were measured with a Vernier caliper every 2-3 days, and tumor volumes

were calculated. On Day 11, when both tumors were palpable, animals were randomly

assigned to 8-groups receiving or not radiotherapy (8 Gy x 3 fractions), to only one of

the two tumors, in combination or not with intraperitoneal immunostimulatory




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monoclonal antibodies (anti-PD1, anti-CD137 or both). Anti-PD1, anti-CD137, the

combination or anti-RatIgG control antibody were administered i.p. at the dose of 200

μg/mouse (10 mg/kg) or 100 μg/mouse (5mg/Kg) on days 13, 15 and 17. In some

experiments monoclonal immunostimulatory antibodies were administered on days 17,

19, 20. Tumor size was monitored every 2-3 days and mice were sacrificed when tumor

size reached 4,000 mm3. (Tumor radiotherapy procedures are detailed in supplementary

materials section).

Flow cytometry and ELISA assays

Tumor tissue was processed to obtain single cell suspension for flow cytometry

analysis (see supplementary methods). To estimate absolute numbers in cell suspension

perfect count microspheres were used as an internal standard according to manufacturer

instructions (Cytognos, Salamanca. spain).

Levels of human IFNγ in mouse plasma samples were measured by a

commercial enzyme linked immunosorbent assay (ELISA; Human IFNγ Elisa Set, BD

OptEIA, BD Biosciences), following the manufacturer's instructions. All samples were

measured in duplicate. The detection cutoff levels of the assay were 4.7 pg/mL for

IFNγ. The coefficient of variation was <15%. For tumor antigen-specific CD8 T-cell

assessment a H-2Kb KSPWFTTL tetramer labelled with PE (manufactured by

Biolegend) were used. For gating and costaining the following mAbs were used CD45.2

PerCP/Cy5.5 (clone 104 from Biolegend), CD4 BV421 (clone RM4-5 from Biolegend),

CD8 BV510 (clone 53-6.7 from Biolegend), CD137 biotin (clone 17B5 from

Biolegend), PD-1 FITC (clone 29F.1A12 from Biolenged).

Statistical analysis




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Statistical differences between survival curves were analyzed with the Mantel-

Cox, log-rank test, non-lineal-regression and differences between other groups were

analyzed with the Mann-Whitney U test using GraphPad Prism (GraphPad Software

Inc., La Jolla, CA)

Results

Abscopal effects of radiotherapy are synergized by anti-CD137 and anti-PD1

immunomodulatory mAb.

Mice bearing bilateral tumors derived from subcutaneous engraftment of MC38

colorectal carcinoma cells were used as a model to monitor the abscopal effects of

radiotherapy in combination with immunostimulatory mAbs. Eight Gy fractionated

doses of external beam radiotherapy were selectively applied only to one of the tumor

lesions, while a contralateral tumor was set outside the irradiation field (see a

representative dosimetry in supple figure 1A). Contralateral concomitant tumors were

inoculated the same day with 10-fold fewer tumor cells. Radiotherapy given every other

day was followed on alternate days by three doses of anti-CD137 or/and anti-PD1

mAbs. The monoclonal antibodies were given as single agents or in combination as

detailed in supple figure 1B. Supplementary Table 1A individually shows the statistical

comparisons of the evolution of irradiated and non-irradiated tumor lesions. Results

collectively indicate that both anti-PD1 and anti-CD137 mAb contributed to control

contralateral tumor growth when in conjunction with unilateral radiotherapy. Strikingly,

the mice receiving radiotherapy and the combination of the two immunostimulatory

monoclonal antibodies were the group that achieved faster and almost constant

complete responses (supple figure 1C), translated in 100% long term overall survival




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(supple figure 1D). Of note, cured mice were immune 3 months later to MC38 tumor

cell rechallenge, while able to engraft B16OVA melanoma cells as an antigenically

unrelated control (supple figure 1E).

Of note, combined treatment was well tolerated by the mice in terms of safety.

Given the fact that CD137 mAb can cause liver inflammation (31), we assessed ALT

serum levels and checked liver pathology specimens that ruled out increased toxicity

due to the addition of local radiotherapy to the immunostimulatory antibody

combination (unpublished observations).

Similar experiments were carried out with bilateral B16OVA melanoma (Figure

1A), known to be of difficult treatment by immunotherapy (32, 33). In this case, mice

bearing tumors for 11 days showed a radiotherapy-dependent control of contralateral

tumors, when distant radiotherapy was combined with either anti-PD1 or anti-CD137

mAb. When both antibodies were combined together all the tumors regressed

bilaterally, even though combined immunotherapy without irradiation also induced the

regression of most tumors (figure 1Band supple Table 1A) achieving long term survival

(figure 1C). When mice cured by the radiotherapy plus mAb combination were

rechallenged 3 months later, 3 out of 5 mice were protected from B16OVA, while

growing MC38 as a contralateral antigenically unrelated control tumor (unpublished

observations). Remarkably, measurements of the concentration of IFNγ in sera from

mice undergoing triple combined treatment (RT plus anti-PD1 plus anti-CD137)

showed on day +18 much higher levels than any other treatment regimen (figure 1D).

This fact strongly indicated an ongoing cellular immune response of far greater

intensity.




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4T1 breast cancer is an exceedingly difficult tumor model to treat with

immunotherapy (34) that causes spontaneous lung metastases. In this setting, we again

performed experiments with bilateral tumors, irradiating only one of the lesions (figure

2A). Tumor growth analyses indicated better local and distant tumor control when

radiotherapy was combined with immunotherapy but without achieving complete

responses (figure 2Band supple Table 1A), although this treatment did lead to longer

survival (unpublished observations). Furthermore, spontaneous metastases to the lung

were followed by CT-scans and by surgical inspection upon sacrifice with

quantification of the number and size of metastases (Figure 2C and supplementary

figure 2). As can be seen in figure 2C overall numbers of spontaneous lung metastases

were reduced in the radiotherapy plus combined mAb immunotherapy group. In supple

figure 2 individual CT-SCAN sections and representative excised lungs are shown.

In the case of bilateral MC38 tumors experiments were also performed starting

treatment as late as day +14 (figure 3A) post tumor cell engraftment to ascertain the

limits of the strategy and demonstrate radiotherapy synergy with the anti-CD137 plus

anti-PD1 combination regimen. In this case, the treatments were not curative in any

case (figure 3B and C), but the delay in tumor progression induced by the triple

combination (RT plus anti-PD1 plus anti-CD137) was readily seen in comparison when

monitoring the contralateral tumor. No noticeable effects were exerted by each of the

mAb when used separately in this regimen, or when the immunotherapy combination

was employed without radiotherapy (figure 3B and C and supple table 1 B).

CD8 T cells, BATF-3-dependent dendritic cells and the type I IFN system are

necessary for the radiotherapy abscopal effects potentiated by anti-CD137 and anti

PD1 mAbs.




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Using treatment conditions comparable to those in figure 1 and supplementary

figure1, on bilateral MC38-derived tumors, we repeatedly depleted CD8β+ T cells, CD4+

T cells and NK 1.1+ lymphocytes with specific monoclonal antibodies (figure 4A). As

can be seen in figure 4B and C in mice treated with the combinatorial regimen of

radiotherapy plus anti-CD137 and anti-PD1 mAbs, we observed that CD8 T cells were

absolutely required for the contralateral antitumor effects. In contrast, CD4 T cell

depletion resulted in a more pronounced therapeutic effect with all animals achieving

complete bilateral regressions and showing a striking effect on overall survival. NK1.1

depletion had little effect on the outcome of the contralateral tumors (figure 4 B and C

and supple table 1C). These results show the involvement of cytolytic T lymphocytes in

the beneficial effect of the combinatorial regimen. CD4 depletion also eliminates

regulatory T cells likely explaining the better outcome upon depletion with anti- CD4

mAb. Induction of CTLs against tumor antigens is mainly mediated by BATF-3-

depended DC (20). Accordingly, we performed similar experiments in BATF-3

deficient mice which showed no abscopal effects and had weaker local tumor control by

radiotherapy (figure 5A and B). In line with this, BATF-3-/- mice showed no increases

in serum IFNγ following combined treatment (Figure 5C)

Since both CTLs (35) and crosspriming (36) are known to be, dependent on type

I IFNs, we next studied treatment in the same experimental setting (Figure 5A) of mice

devoid or not of type I IFN receptor (IFNAR-/-). As can be observed in figure 5D, the

abscopal effects exerted by combined radioimmunotherapy were completely abrogated

in IFNAR-deficient mice. Importantly, the local effects on the directly irradiated tumors

were also decreased to same extent, suggesting an important role of the IFNα/ ß system

on the therapeutic effects exerted by radiotherapy.




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Radiotherapy changes the immune tumor microenvironment in non-irradiated

tumor lesions

Observations of therapeutic effects on tumor lesions outside the irradiation fields

prompted us carry out experiments to investigate changes in the immune contexture of

the tumor microenvironment due to irradiation. Our 8 Gy fractionated doses on alternate

days (scheme in supple figure 3A) was applied to mice bearing bilateral MC38-derived

tumors. Absolute numbers and density of T lymphocyte subsets were quantitated at the

end of the regimen on day +17. We observed that CD4 T cell and CD8 T cell numbers

were clearly reduced both at the tumor site receiving radiation and, importantly, at the

tumor lesion outside the irradiation field. FOXP3+ CD4+ T reg cells were also reduced

at the contralateral site and less clearly so at the irradiated site (supple figures 3 B and

C). Myeloid-delivered suppressor cells were also evaluated as CD11b (Ly6C or Ly6G)

positive cells in the tumors. Our results indicate a trend towards a decrease in G-MDSC

in the irradiated tumor, whereas in the non-irradiated sites M-MDSC were decreased to

some extent (supple figure D and E). However, our repeated experiments did not reach

statistical significance.

The results on radiotherapy-dependent reduction of tumor infiltrating T

lymphocytes were confirmed in mice bearing MC38 tumors in which abscopal effects

were noted (Supple figure 4 A to C). However in this case combined radiotherapy plus

combined immunotherapy gave rise to dramatic increases of CD4 and CD8 T cells

infiltrating the irradiated and distant tumors (supplementary figure 4C).

Moreover, analyses with a MHC tetramer that detects specific CD8 T cells

recognizing the gp70 immunodominant antigen in MC38 tumor cells showed a clear

tendency to higher numbers of such tumor-reactive CD8 T-lymphocytes in the tumor




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microenvironment observed in mice undergoing combined treatment (supple figures 4

D and E).

It was also important to assess functionality in terms of the ability of tumor-

infiltrating T lymphocytes to produce IFNγ. To this end, we treated mice as in supple

figure 1 (figure 6A) and mice were sacrificed on day +16 to monitor tumor-infiltrating

T cells. In this setting, experimental groups undergoing combined treatment showed

smaller tumor lesions on both sides (figure 6B). Intratumoral CD4 and CD8-gated T

cells were assessed for the intensity of intracellular IFNγ staining with further

stimulation ex-vivo with PMA and ionomycin. As seen in figure 6C and D,

lymphocytes from mice undergoing combined radiotherapy plus immunotherapy

attained more intense IFNγ production both in lymphocytes from the irradiated and non-

irradiated lesions. Of note these differences were also observed if the lymphocytes were

not stimulated with PMA plus ION (unpublished observations).

Radiotherapy enhances the expression of CD137 and PD1 on tumor infiltrating

lymphocytes

One possible explanation of the synergy between radiotherapy and

immunostimulatory monoclonal antibodies was that radiotherapy resulted in a more

intense expression of CD137 and/or PD1 on tumor infiltrating T lymphocytes. Using

multicolor immunofluorescence and flow cytometry an increase of CD137 expression

levels was observed among TILs 48 hours following a 20Gy single dose (Supple figure

5A ). Such an effect was more conspicuous on CD8+ TILs, while PD1 expression was

preserved at a similar bright level as in the case of the non-irradiated tumor. PD-L1 was

expressed on TILs with slight increases related to radiotherapy. On TILs in the

contralateral non-irradiated lesion, CD137 levels also increased on CD8 T cells but not




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on CD4 T cells. Increases in PD1 were also noted but only on CD8 T cells in the

contralateral side (supple figure 5A and B). This was not observed when only a single

dose of radiotherapy was given, since following three fractionated 8 Gy doses an

increase in the expression of PD-1 and CD137 was also documented (Supple figure 5C

and D).

To study these effects on human tumor samples, we irradiated freshly surgically

explanted adenocarcinomas (two gastric carcinomas, five colon cancers and one

condrosarcoma). Fragments of the excised tumor received 20 Gy, while the other

fragments were left without irradiation (mock irradiated). Tumors fragments were

subsequently maintained in culture medium and 48 hour later, samples were formalin-

fixed and paraffin-embedded for immunohistochemical analysis. As can be seen in

supplementary figure 6A and B, there was a clear increase in the percentage of TILs

with CD137 and PD1 detectable surface expression, while the total number of T cells

remained without noticeable changes. Supplementary Figure 6B shows

microphotographs of representative immunohistochemistry fields of one of the cases.

To study these increased expression of CD137 and PD-1 at the single cell level in a

more quantitative fashion flow cytometry analyses were performed in two cases of

colon cancer as freshly surgically excised tumors. Figure 7A shows clear increases in

immunofluorescence intensity for PD-1 and CD137 on viable CD8 or CD4 T cells that

was contingent upon irradiation. In figure 7B, dot plots of these cases show that CD8

and CD4 T cells frequently co-expressed CD137 and PD-1 following irradiation.

Furthermore, multiplex tissue immunofluorescence on a surgical specimen of gastric

cancer showed augmented expression of CD137 and PD-L1 upon irradiation and

representative images are shown in figure 7C.




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All in all, radiotherapy-induced increases in expression of the mAb-targeted

receptors PD1 and CD137 are thus likely to account, at least in part, for the

combinatorial synergistic effect of radiotherapy and infusion of immunomodulatory

mAb targeted to such receptors.




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DISCUSSION

Cancer therapeutics are likely to benefit from the combination of radiotherapy

and immunotherapy strategies (13). Our study also strongly indicates that radiotherapy

can become a treatment with systemic beneficial antitumor effects (abscopal effects), in

addition to the well-known local effects of irradiation. In our hands radiotherapy

modifies the immune microenvironment of distant non-irradiated tumors, but only the

addition of immunostimulatory monoclonal antibodies was able to elicit a meaningful

therapeutic effect against non-irradiated tumors and consolidate or enhance the response

against the directly irradiated malignant lesions.

To explain the abscopal effects of radiotherapy many mechanisms have been

invoked. Radiotherapy causes vascular inflammation (13) and activation of antigen-

presenting dendritic cells (37). Recently the key contribution of sensing tumor released

DNA by the cytoplasmatic pattern recognition receptor STING was found to be

crucially important. This mechanism critically causes a local release of type I IFN

involving dendritic cells (36). Irradiation is also reported to kill tumor cells showing the

hallmarks of immunogenic cell death, as defined by Kroemer´s and Zitvogel´s groups

(38). In this study we find that abscopal effects are contingent on a dendritic cell subset

specialized in antigen crosspriming to induce CTLs (20). It is tempting to speculate that

such antigen presenting cell subset is the main mediator of productive tumor antigen

presentation to CD8 T cells.

CTL responses and crosspriming are known to be dependent on type I IFN in

mice (36, 38). This cytokine system has evolved to raise the alarm upon acute viral

infection and is involved in setting in action an optimal immune response for viral

clearance. Our findings demonstrate that IFNα/ß is critically involved in the abscopal




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effects of radiotherapy. DNA released form dying tumor cells is probably involved in

eliciting IFNα/ ß via STING (39) and, in turn, IFNα/ß may act both on crosspriming

dendritic cells (36) and of CD8 T cells (35) to favor, as a necessary factor, the CTL

immune response. Strategies aiming at local enhancement of IFNα/ß could render

radiotherapy-induced tumor cell death more immunogenic as recently shown for

chemotherapy (40).

However, immunogenic cell death as induced by radiotherapy only

exceptionally offers systemic control of the spread of disease. This could be due to the

relative weakness of the immunizing effects or because of concomitantly elicited

immunosupressor factors and mechanisms such as those mediated by TGFβ (41).

Strategies to enhance the antitumor immune effects of radiotherapy have been

explored in preclinical models, and results from pioneering clinical research have also

been reported (28). For instance, mouse tumor lesions were treated with the TLR7

agonist imiquimod cream (42, 43), or injected with TLR9 CpG agonist nucleotides

showing evidence for stronger immunity with the ability to partially tackle distant

disease (44, 45). In the clinic strategies based on combinations of radiotherapy with

imiquimod (42) or subcutaneous GM-CSF (46) have been reported with promising

proof-of-concept results.

Regarding the optimal combinations of radiotherapy and immunotherapy,

several parameters are to be optimized including dose, fractionation and interval

between doses. We chose three fractions of 8 Gy based on published evidence (47)

suggesting that this regimen attains better results from the immunological point of view

at least when combining radiotherapy with anti-CTLA-4 mAb (48). However, this issue

remains open to debate.




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Monoclonal antibodies that tamper with immune inhibitory receptors

(checkpoints) (2) or agonist antibodies to lymphocyte costimulatory receptors (49) have

taken the center stage of oncology drug development. A plethora of clinical trials are

exploring their efficacy against multiple malignant diseases, first when used as single

agents and then in combinations(50). To date, very little clinical experience exists with

combining radiotherapy with immunostimulatory monoclonal antibodies. Only results

from two clinical trials combining radiotherapy and ipilimumab are available for

metastatic melanoma (28) and prostate cancer (51). These showed limited efficacy that

might be patient subset-specific. As for abscopal effects, evidence is even more scanty

although there is reported anecdotal evidence (30).

Systemic effects of anti-PD1 mAb have not yet been reported to potentiate

abscopal effects of radiotherapy in patients. In immunogenic mouse models, anti-

CD137 (25) and anti-PD1 mAb (21) have been reported to enhance the antitumor

effects of radiotherapy. In our case we report that these antibodies, and especially their

combination, can unleash a very potent therapeutic effect against the contralateral

tumors (abscopal effects), when both irradiated and non-irradiated tumor lesions were

very well established for longer than one week.

In our experiments, potentiation of abscopal effects resulted in long-term

survival, comparable to recently reported effects with the combination of anti-PD1 and

anti-CTLA-4 mAb in B16F10-bearing mice (28). In this case, the combined mechanism

resulted from a reinvigoration of antitumor CTLs that were not repressed by the

PD1/PD-L1 axis if combined treatment was given. Our selective depletion experiments

point in the same direction.




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Our results on abscopal effects contrast with the fact that radiotherapy by itself

reduced in our hands the content of T cells in the tumors, although it also slightly

reduced the number of MDSC both in the irradiated tumor and in the contralateral site.

However, radiotherapy enhanced IFNγ production on a per cell basis and the level of

CD137 and PD1 expression on T cells, in this way making them more amenable to

pharmacological therapeutic costimulation. This was also observed in human tumor

fragments irradiated ex-vivo. Interestingly our depletion experiments reveal that only

the function of CD8+ T cells is an absolute requirement. Moreover, at least a subset of

CD4+ cells seems to be operating in detriment of efficacy. These are likely to be Treg

cells. Importantly, at the single cell level there are tumor infiltrating lymphocytes that

coexpress the CD137 and PD-1 receptors, arguing in favour of a double hit by the

immunostimulatory mAbs on single T-cell basis.

In our hands, radiotherapy plus immunostimulatory monoclonal antibodies

dramatically enhance the T-cell infiltrate following 8 days of combined treatment with

evidence for more CD8 T cells recognizing the gp70 tumor antigen in the MC38 tumor

model. These tetramer-positive cells are CD137+ and PD-1+ in keeping with previous

reports showing that CD137+ cells in human melanomas tend to be specific for tumor

neoantigens (52).

Overall, our data strongly support initiation of clinical trials testing anti-CD137

mAb in combination with PD1/PD-L1 blockade together with concomitant irradiation

of some of the tumor metastatic sites, in search of a way powerful to make the most of

these novel immunotherapies.




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Acknowledgments: We acknowledge generous help by Drs. Martinez-Monge,

Aristu, and Gil-Bazo from the department of oncology at CUN. We are also grateful for

the advice from Drs. Lozano, Echeveste and Idoate from the pathology department at

CUN. We are grateful for Sciencific discussion with Drs Mariano Ponz, David Sancho,

Nicola Tinari and Antonio Rullán. Excellent Dosimetry by Arantza Zubiria and

dedicated animal care by Eneko Elizalde are also acknowledged.




22

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Figures Legends

Figure 1: Combined radiotherapy and immunotherapy with anti-CD137

and/or anti-PD1 mAbs against B16OVA derived tumors. (A) scheme of tumor

engraftment and combined treatments. (B) Bilateral tumor size follow-up (mean) of the

indicated treatment regimes (only the primary tumors received radiotherapy when

indicated) Supple Table 1A shows statistical comparisons. (C) Overall survival in the

same groups of mice. (D) Serum concentrations of IFNγ pretreatment and 48h after

completing the regimen of the indicated mAbs with or without prior radiotherapy (RT)

dose of 8Gy x 3 fractions.

Figure 2: Combined radiotherapy and immunotherapy with anti-CD137

and/or anti-PD1 mAb mediate combined effects against 4T1-derived breast

carcinomas and reduction of spontaneous lung metastases. (A) Treatment scheme and

follow-up (B) subcutaneous tumor growth follow-up (mean) of the primary and

contralateral tumors. Only the primary tumor received radiotherapy when indicated

(Supple Table 1A shows statistical comparisons). (C) Number of lung metastasis

identified by CT-SCAN on day +36 (mean±SD) in the indicated treatment groups.

Representative data are shown in suppl figure 2 including photographs with the

spontaneous metastases seen in mice whose lungs were excised upon necropsy.

Figure 3: Delayed treatment of bilateral MC38-derived tumors shows

synergistic effects combining immunotherapy with anti-CD137 plus anti-PD1 mAbs

with radiotherapy. (A) Scheme of treatment as in supplementary figure 1 but delaying

treatment onset until day +14. As in previous figures, only the primary tumors received

radiotherapy when indicated. (B) Average bilateral tumor progression in the indicated

treatment groups. (C) Follow-up of individual bilateral subcutaneous tumors from (B).

Statistical comparisons by non-linear-regression are shown in Supple table 1B.




27

Figure 4: CD8+ T cells are necessary for immune-mediated abscopal effects of

radiotherapy potentiated with the combination of anti-CD137 and anti-PD1 mAbs.

(A) Scheme of treatments and depletions of CD4, CD8 and NK lymphocytes with specific

antibodies.

(B) Follow-up of the growth of subcutaneous MC38-derived tumors as bilaterally

implanted (again only the primary tumors received the doses of radiotherapy when

indicated). (C) Overall survival. Statistical comparisions are shown in supple table 1C.

Figure 5: BATF-3-/- and IFNAR-/- mice lose the abscopal effects of

radiotherapy upon combination treatment with anti-PD1 and anti-CD137 mAbs. (A)

control or combined immunotherapy plus radiotherapy treatment regimens were given as

in supplementary figure 1 to C57Bl6 WT mice or to syngenic BATF3-/- or IFNAR-/-

Mice. (B) Shows the average tumor growth in the directly irradiated tumor lesion and the

contralateral tumor in BATF-3-/- mice in comparison with WT mice. (C) Concentrations

of IFNγ in the serum samples from the indicated groups of mice taken 48 hours following

the last treatment dose. (D) Similar experiment as in B comparing IFNAR-/- mice with

WT mice.

Figure 6: Combined immunostimulatory monoclonal antibodies and unilateral

radiotherapy induce more intense expression of IFNγ in CD8+ and CD4+ tumor

infiltrating T cells. (A) scheme of treatment and tumor surgical excision in mice bearing

bilateral MC38-derived tumors. (B) tumor weight at sacrifice (day +16). (C) Mean

fluorescence intensity (MFI) of intracellular IFNγ immunostaining in the gated CD8+ or

CD4+ tumor infiltrating T lymphocytes as indicated following a 4 hour re-stimulation

with PMA + ionomycin. (D) Percentage of lymphocytes expressing intracellular IFNγ

among CD8+ and CD4+ tumor-infiltrating lymphocytes.




28

Figure 7: Ex-vivo irradiation induces CD137, PD-L1 and PD1 expression in

human carcinoma tissue samples. (A) Two primary human colon carcinomas were

surgically excised and following pathology assessment, tumor fragments were minced to 5

x 5 mm pieces and kept in tissue culture. Samples were irradiated or non-irradiated (mock-

irradiated) with a single dose of 20 Gy and 48 hours later cell suspensions were

immunostained for flow cytometry and fluorescence intensity of immunostainings for

surface CD137 and PD-1 on gated CD4 and CD8 T lymphocytes are shown. (B) Dot plots

show the percentages of double positive lymphocytes for CD137 and PD-1 on CD4 and

CD8 T cells in the tumor-derived cell suspensions corresponding to irradiated and mock-

irradiated tissue samples as indicated. (C) Multiplexed immunofluorescence

microphotographs of a representative gastric carcinoma explant whose fragments were

either irradiated or mock irradiated, showing stainings for tumor cells (cytokeratin-

positive, green channel), CD3 (green chanel), CD137 or PD-L1 (red channels). Nuclei

were highlighted with DAPI (blue fluorescence). Bar= 100 um.

























Published OnlineFirst August 22, 2016.Cancer Res Maria E. Rodriguez-Ruiz, Inmaculada Rodriguez, Saray Garasa, et al. and crosspriming.immunostimulatory mAbs and are dependent on CD8 T cells Abscopal effects of radiotherapy are enhanced by combined

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María E. Rodriguez-Ruiz 1,2 1 - Cancer Researchcancerres.aacrjournals.org/content/canres/early/2016/08/20/0008... · 1 Abscopal effects of radiotherapy are enhanced by combined immunostimulatory