Summary

Results

PD-L1-specificTcellsareactivatedbyinflammationandcanbereadilyexpandedbyepitopevaccination

MiaThorupLundsager1,2,MadsHald Andersen2,11IOBiotechApS,Copenhagen,Denmark

2CenterforCancerImmuneTherapy,DepartmentofHematology,Herlev Hospital,Herlev,Denmark

Background. Checkpoint inhibitory pathways, such as the programmed death-1 receptor (PD-1) and its ligand (PD-L1), play key roles in inducing immune tolerance in thetumor microenvironment. PD-L1 is expressed in the tumor microenvironment by cancer cells as well as various immune regulatory cells, like dendritic cells. We have previouslydemonstrated that the immune system has an anti-cancer mechanism that works via PD-L1-specific effector T cells in vitro1,2: thus, detectable populations of CD8+ and CD4+ Tcells specific to PD-L1 were found in peripheral blood of healthy donors and patients with various cancers. The PD-L1-specific T cells exhibit cytotoxic function and are capable ofkilling PD-L1 expressing melanoma cells. This led us to hypothesize that PD-L1-specific T are present at steady state and may have a role in modulating adaptive immunesuppression. Targeting such T cells by vaccination may lead to elimination of PD-L1 expressing tumor cells as well as the PD-L1 expressing regulatory immune cells, therebypotentiating effective anti-tumor immune responses. The present study aims at establishing a translational preclinical model to evaluate this in C57BL/6 mice.

Results. A computer algorithm was employed to predict long (18+ aa) murine MHC-restricted PD-L1 epitopes and the most immunogenic peptide, mPD-L1long (18aa) waschosen based on the frequency of PD-L1-specific T cell responses [defined by IFNγ-releasing cells with a specificity towards mPD-L1long] by IFNγ-Elispot in spleens from miceinjected with IFNγ two times.1. Inflammation-mediated expansion of PD-L1 specific T cells: To assess the effect of inflammation on PD-L1-specific T cells, C57Bl/6 mice were either treated with IFNγ i.p.

(Figure 1) or with an allergen, 2,4-dinitrofluorobenzene (DNFB) on the ears (Figure 2), and the frequency of PD-L1-specific T cells was analyzed by Elispot. The expansion ofPD-L1 specific T cells was detected in the spleen and dLNs after two IFNγ-injections or 3 treatments with DNFB, respectively, indicating a rapid increase in PD-L1-specific Tcells.

2. Expansion of PD-L1-specific T cells by mPD-L1long vaccination: C57Bl/6 mice were vaccinated s.c. with mPD-L1long in Montanide to activate the PD-L1-specific T cells.The frequency of PD-L1-specific T cells was assessed by Elispot and intracellular cytokine staining (ICS) (Figure 3). After single vaccination with mPD-L1long it was possible toexpand the frequency of PD-L1-specific T cells. These PD-L1-specific T cells were shown to be CD8+ T cells.

References1Munir et al. HLA-Restricted CTL That Are Specific for Immune Checkpoint Ligand PD-L1 Occur with High Frequency in Cancer Patients, Cancer Research 2013;73:1764-1776.2Munir et al. The immune checkpoint regulator PD-L1 is a specific target for naturally occurring CD4+ T cells, OncoImmunology 2013;2:e23991.

Expansion of PD-L1-specific T cells after mPD-L1long vaccination

Figure 3. One vaccination with mPD-L1long expands the population of PD-L1-specific T cells. Thispopulation is found to be CD8+ T cells. C57Bl/6 mice were vaccinated s.c. with Montanide or mPD-L1long/Montanide at day 0 (A, timeline). At day 7 the mice were sacrificed and spleen and dLNs analyzedby IFNγ-Elispot (B,C) and Intracellular cytokine staining (ICS) (D) with or without ex vivo stimulation withmPD-L1long. n=12-13 mice/group

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Figure 1. To show a natural PD-L1-specific T cellresponse, C57Bl/6 mice were injected with IFNγi.p. two times two days apart to induceinflammation (A, timeline). At day 6 the mice weresacrificed and spleens removed for furtheranalysis by IFNγ-Elispot with or without ex vivostimulation with mPD-L1long (B,C).n=5-10 mice/group

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Mia Thorup LundsagerIndustrial PhD studentMia.thorup.lundsager@regionh.dk

IO Biotech, Denmark,http://www.iobiotech.comCenter for Cancer Immune Therapy,Herlev Hospital, Denmark, https://www.herlevhospital.dk/ccit-denmark/Sider/default.aspx

PD-L1-specific T cell response by local inflammation

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Figure 2. 2,4-dinitrofluorobenzene (DNFB) is anallergen that induces local inflammation. Here thismodel is used to show that PD-L1-specific T cellsare activated by inflammation. DNFB were paintedon the dorsal side of both ears of C57Bl/6 mice on3 consecutive days (A, timeline). On day 5 themice were sacrificed and spleens and dLNsremoved for further analysis by IFNγ-Elispot withor without ex vivo stimulation with mPD-L1long(B,C). N=12 mice/group

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We describe that PD-L1-specific T cells are expanded by IFNγ-injections and by inflammation (DNFB), suggesting that PD-L1-specific Tcells are already present and are activated due to a strong activation signal from their cognate targets (i.e. professional antigen-presenting cells) at inflammation sites. PD-L1-specific T cells are readily expanded by vaccination.Thus, PD-L1-specific T cells are a particularly interesting example of the immune system’s ability to influence adaptive immuneresponses by directly reacting against the immune-suppressive mechanisms employed by cancer cells.

The next step is to investigate the anti-tumor effect of the mPD-L1 vaccine. Preliminary studies investigating the potential of a PD-L1 vaccine in a B16 melanoma model have shown indications of an anti-tumor effect in vivo. This is currently evaluated in other murine tumor models.

Conclusion

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