Blockade of TGF-β and PD-L1 by bintrafusp alfa promotes survival in preclinical ovarian cancer models by promoting T effector and NK cell responses

Background Failure of immunotherapy in high-grade serous ovarian cancer (HGSC) may be due to high levels of transforming growth factor-β (TGF-β) in ascites or tumour immune microenvironment (TIME). Here, we test whether coordinated blockade of TGF-β and PD-L1 with bintrafusp alfa (BA) can provoke anti-tumour immune responses in preclinical HGSC models. Methods BA is a first-in-class bifunctional inhibitor of TGF-β and PD-L1, and was tested for effects on overall survival and altered TIME in syngeneic HGSC models. Results Using a mouse ID8-derived HGSC syngeneic model with IFNγ-inducible PD-L1 expression, BA treatments significantly reduced ascites development and tumour burden. BA treatments depleted TGF-β and VEGF in ascites, and skewed the TIME towards cytotoxicity compared to control. In the BR5 HGSC syngeneic model, BA treatments increased tumour-infiltrating CD8 T cells with effector memory and cytotoxic markers, as well as cytolytic NK cells. Extended BA treatments in the BR5 model produced ∼50% BA-cured mice that were protected from re-challenge. These BA-cured mice had increased peritoneal T-effector memory and NK cells compared to controls. Conclusions Our preclinical studies of BA in advanced ovarian cancer models support further testing of BA as an improved immunotherapy option for patients with advanced ovarian cancer.

BR5-Luc cells (4x10 6 ) were injected into female FVB mice randomized between Control IgG (400 µg), TGF-β Trap (492 µg), anti-PD-L1 (400 µg) or BA (492 µg).Treatments were administered 3 times, 2 days apart during week 7 to avoid neutralizing antibodies produced by B cells that limit efficacy of testing bintrafusp alfa.At endpoint, cells from dissociated tumors were subject to flow cytometry for immunophenotypic analysis of a T cells using antibodies targeting CD4, CD44, CD62L, along with intracellular markers for IFN-γ and FOXP3.b NK cells were classified using antibodies targeting surface markers for CD49b, CD107a, CD27 and CD11b (* p<0.05, ** p<0.01, *** p<0.001 based on ANOVA with multiple comparison testing).Overall, 30,000 lymphocytes were recorded, and data was exported as %parent normalized to %live CD45 + immune cells (see S7 for gating strategies).At endpoint, tumors were mechanically digested prior to staining with flow cytometry.a Representative gates for non-debris/lymphocytes, singlets and live CD45 + immune cells were applied as baseline gates for all antibody cocktails.b CD8b + T cells were further classified with antibodies targeting surface markers for PD-1, CD44, CD62L, and IFN-γ in a separate intracellular cocktail.Similarly, CD4 + T cells were further classified with antibodies targeting surface antigens for CD44, CD62L, along with FOXP3 and IFN-γ in separate intracellular cocktails.c Last, CD49b + NK cells were further classified with antibodies targeting surface markers for CD27, CD11b and CD107a.Overall, 30,000 lymphocytes were recorded, and data was exported as %parent normalized to %live CD45 + immune cells.Positive populations are denoted by gates on each histogram and * denotes which population each histogram was gated on.Spleens and ascites were harvested from tumor-bearing FVB mice that met protocol-defined endpoints in the extended survival study two weeks after administration of anti-CD20 or control mice without B cell depletion.Splenocytes and ascites cells were stained with fluorophoreconjugated antibodies against CD19 and B220 (along with isotype controls) to compare B cell populations following analysis by flow cytometry.Representative histograms and gating are shown.

Fig. S2
Fig. S2 Altered gene expression patterns in metastatic tumor nodules treated with BA.RNA isolated from metastatic ID8-DKO tumor nodules from the indicated treatment groups was obtained at endpoint and subjected to gene expression analysis using the Nanostring PanCancer Immune Profiling Gene Expression Panel.Analysis of DEGs was performed using nSolver software (Nanostring).a A volcano plot of DEGs in BA-treated tumors compared to tumors treated with Control IgG is shown (* p<0.05, ** p<0.01, *** p<0.001 based on ANOVA with multiple comparison testing).b/c Box plots are shown comparing Thbs1 or exhausted T cell signatures between control IgG and BA (p<0.05 based on unpaired T-test).

Fig. S4
Fig. S4 Characterization of the syngeneic BR5-Luc mouse HGSC for testing of BA. a Starved BR5-Luc cells were co-treated with TGF-β1 (5 ng/uL) and increasing doses of TGFBR1 inhibitor LY2157299/Galunisertib (0.1-10 µM) for one hour and Smad2/3 phosphorylation was compared using immunoblotting.b BR5-Luc cells were stimulated with TGF-β1 (5 ng/uL) or IFNγ (20 ng/uL) for 72 hours and surface expression of PD-L1 was compared using flow cytometry.c The graph depicts a quantification of the percentage of cells expressing PD-L1 (* p<0.05 based on unpaired T-test).

Fig
Fig. S5 Limited treatment window with BA showed promising effects in immunocompetent FVB mice.a BR5-Luc cells were injected into the peritoneum of female FVB mice.b After 7 days, weekly IVIS imaging was performed to measure luminescence as a readout for HGSC tumor burden as described in Materials and Methods.On days 11, 33 and 15, mice received i.p. injections of BA (492 μg) or a control IgG (400 μg; n=10/group).c Graph depicts luminescence quantification as total flux for each treatment group over the indicated time points (* p<0.05).d Kaplan-Meier survival curve was generated for animals that reached animal protocol-defined humane endpoints.

Fig. S6
Fig. S6 Assessment of TIME with short term bintrafusp alfa treatments in BR5 syngenieic model.

Fig. S7
Fig. S7 Representative flow cytometry gating strategies using antibodies targeting surface and intracellular antigens on T/NK cells isolated from dissociated tumors in the BR5-Luc endpoint study.

Fig. S8
Fig. S8 Extent of B cell-depletion in spleen and ascites of FVB mice 2 weeks after Anti-CD20 injection.

SUPPLEMENTARY MATERIALS SUPPLEMENTARY TABLES Supplementary Table 1. Significantly upregulated genes in BA vs IgG-treated HGSC tumors
¥ employed during time of the study