Targeting the TGFβ pathway with galunisertib, a TGFβRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade

Targeting the TGFβ pathway with galunisertib, a TGFβRI small molecule inhibitor, promotes... Background: TGFβ signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFβ’s immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFβ pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses. Results: In vitro treatment with galunisertib reversed TGFβ and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti- tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFβ and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy. Conclusions: Together these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy. Keywords: TGF-β receptor I, Antitumor efficacy, Checkpoint inhibitors, Galunisertib * Correspondence: Driscoll_kyla@lilly.com Rikke B. Holmgaard and David Schaer contributed equally to this work. Eli Lilly and Company, 450 East 29th Street, New York, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 2 of 15 Background progression and anti-tumor immunity. That TGFβ may be Transforming growth factor-beta (TGFβ) has been iden- involved in the maintenance of self-tolerance and patho- tified as a therapeutic target in cancer because of its sig- genesis of systemic inflammatory diseases is indicated in nificant and varied roles to promote tumor growth, studies which show the development of multi-organ in- −/− survival, and metastasis. There are several pharmaco- flammation in Tgfb1 mice [15, 16]. The inflammation −/− logical approaches to block TGFβ signaling, including in Tgfb1 mice is dependent on T cells, which undergo neutralizing antibodies, vaccines, antisense oligonucleo- massive activation [17]. Generation of mice lacking tides and small molecular inhibitors (SMI) [1, 2]. The TGFβRII specifically on T-cells further demonstrates the goal of these therapies is to block the tumor-promoting importance of TGFβ in regulating T-cell responses in vivo, effects of TGFβ, while maintaining its tumor suppressive as mice develop multi-organ inflammation similar to that properties. Emerging data and thought suggest that the seen in TGFβ1−/− mice [12, 18]. efficacy of TGFβ antagonist therapy in cancer might not In addition to the direct effects on effector T cell only derive from direct intrinsic effects on tumor cells, responses, TGFβ can promote immunosuppression via but also involves tumor extrinsic mechanisms acting in direct induction and modulation of regulatory T cells the tumor micro-environment. (Tregs) [19]. TGFβ directly promotes expression of TGFβ plays pleiotropic roles to initiate and progress Foxp3 in CD4 T-cells, converting them to a regulatory cancer including both tumor cell intrinsic and extrinsic phenotype [20]. In addition to induction and mainten- activities. Tumor cell intrinsic activities of the TGFβ ance of Foxp3 expression, TGFβ has also been shown to pathway include autocrine TGFβ driven tumor cell pro- be important in the functional ability of Tregs to sup- liferation and differentiation, epithelial to mesenchymal press immune responses [21, 22], and it has been dem- transition (EMT), invasion and migration, prometastatic onstrated that Tgfb1−/− mice fail to maintain peripheral cytokine production, and autocrine mitogen production Treg cells [21]. TGFβ1-producing myeloid-derived sup- [3, 4]. Tumor cell extrinsic activities include promoting pressor cells (MDSCs) have also been reported at high of increased tumor vascularization, modulation of the levels in the tumor microenvironment [23, 24]. stromal extracellular matrix, induction of and feedback Clinical studies have provided proof of concept data modulation of the hypoxic state and inhibition of supporting the role of TGFβ in cancer and the utility of immune surveillance and antitumor immunity [4, 5]. targeting the TGFβ pathway [1]. Galunisertib (LY2157299 Systemic TGFβ ligand levels are often elevated in can- monohydrate) is an oral small molecule inhibitor (SMI) of cer patients compared to healthy individuals, and in- the TGFβ receptor I (TGFβRI) kinase that specifically creased ligand levels have been further associated with downregulates the phosphorylation of SMAD2, abrogating aggressive disease and poor prognosis [6, 7]. Elevated activation of the canonical pathway [1] (Yingling et al., TGFβ ligand levels are observed in patients whose [25]). By targeting TGFβRI, signaling via all three TGFβ tumor cells are both sensitive (i.e. receptor positive, ligands is blocked [1]. Galunisertib demonstrates the abil- TGFβ ligand dependent) or insensitive (i.e receptor ity to inhibit TGFβ-dependent tumor cell intrinsic and negative, TGFβ ligand independent) to TGFβ signaling. extrinsic functions in vitro and in vivo, and to inhibit Furthermore, aberrant TGFβ signaling has been impli- tumor-cell growth in established tumor mouse models cated in several human diseases, including malignancies (Yingling et al., [25]). Galunisertib is currently under clin- such as glioblastoma and breast cancer [8–10]. ical development in combination with checkpoint inhibi- TGFb additionally plays a non-redundant, crucial role tors (including nivolumab and durvalumab) in patients in regulating immunity. TGFβ is produced by a number with NSCLC, HCC, or pancreatic cancer (NCT02423343; of immune cells and plays an essential role in the regula- NCT02734160). tion of immune responses and immune tolerance [4, 11]. In the current study, we set out to characterize in Genetic deletion and antibody neutralization studies detail the impact of galunisertib-mediated TGFβR1 have demonstrated that TGFβ inhibition enhances T cell blockade on anti-tumor immunity. Using both in vitro [12] and NK cell differentiation and function [13], sug- and in vivo model systems, we show that galunisertib gesting that pharmacologic inhibition of TGFβ signaling enhances the development of anti-tumor T cell immun- might decrease the suppression of host immune surveil- ity through modulating both effector and regulatory T lance. Furthermore, deletion of TGFβ signaling in myeloid cell function. Using an immunogenic 4 T1-LP breast cells has been shown to enhance their anti-tumorigenic tumor model, we show that galunisertib mediates robust properties [14]. The immunological consequences of anti-tumor T cell immunity and promotes the establish- TGFβ antagonism are particularly relevant in the context ment of T cell memory and antigen spreading. Using in of anti-tumor immunotherapy, and blockade of the TGFβ vitro assays and primary human Treg cells we show that pathway has become an attractive approach to inhibit the Galunisertib treatment blocks the suppressive activity of multitude effects the TGFβ pathway has on cancer human Tregs, further highlighting its important role in Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 3 of 15 T cell immunity. The TGFβ pathway was recently de- after 5–7 days of culture. Rescue of proliferation was scribed as a potential mechanism of resistance for anti- calculated according to the formula: Percent recovery PD-1/L1 checkpoint blockade [26, 27]. To this end, we of proliferation = (%CFSE low T naive in treated Treg show that galunisertib treatment at a clinically relevant co-culture - %CFSE low T naive in untreated Treg dose enhances the anti-tumor activity of anti-PD-L1 co-culture)/(% CFSE low untreated T naive monocul- resulting in robust tumor regressions associated with en- ture stimulated with beads - %CFSE low T naive in hanced T-cell activation signatures, further supporting untreated Treg co-culture) × 100%. One-way ANOVA the clinical development of targeting TGFβRI in com- followed by Dunnett’s test was performed to assess bination with checkpoint blockade. Clinical trials evalu- statistical significance. ating galunisertib in combination with anti-PD-1 immunotherapy are currently being conducted (https:// Murine cell lines clinicaltrials.gov; NCT02734160 and NCT02423343) and CT26.WT (CT26) colon and 4 T1 and EMT6 breast thus, gives this research a highly translational impact. tumor lines, were purchased from American Type Cul- ture Collection (ATCC; Manassas, VA). MC38 colon Methods tumor cell line was purchased from the NCI tumor re- Human CD8 T cell suppression assays with TGFβ pository (Frederick, MD). The 4T1 luciferase positive CD8 T cells were purified from healthy donor blood (4T1-LP) cell line was developed at Lilly NYC from the (New York Blood Center, NY, NY) with RosetteSep 4T1 parental cell line stably transduced with firefly lucif- Human CD8 T cells enrichment kit (Stemcell erase (luciferase plasmid pLXSN-luc, G418). The EMT- Technologies) and labeled with 1 mM CFSE (Invitrogen) 6-LM2 was generated following serial passage of meta- in pre-warmed PBS+5%FCS for 10 min at 37 °C. Cells static parental EMT6 cells [13]. were then plated onto 96-well plates (5 × 10 /well) in complete RPMI media (Gibco) and stimulated with Mice −/− human T cell activation/expansion beads (Miltenyi Female Balb/c (WT and Rag )and C57BL/6mice(6to Biotech). Cells were cultured with or without TGFβ1at 8 weeks of age) were purchased from Harlan Laboratories/ 10 ng/ml. Galunisertib was added at indicated Envigo. All experimental procedures were done in concentration (0.1μMto10 μM) with DMSO as vehicle accordance with the guidelines of the NIH “Guide for Care control. Percent CD8 T cell proliferation was measured and Use of Animal” and approved protocols reviewed by by assessing CFSE dilution by FACS (BD LSRFortessa) Institutional Animal Care and Use Committee. after 5 days of culture. Recovery of T cell proliferation was calculated according to the formula: % of Max In vivo studies: Tumor challenge and treatment proliferation = % CFSE low of sample/(average of CFSE experiments low for control with no TGFβ). One-way ANOVA 4T1 and 4T1-LP tumors were generated by injection of followed by Dunnett’s test was performed to assess stat- 1×10 cells orthotopically in the mammary fat pad of istical significance. Balb/C mice. Galunisertib was dosed P.O. at 37.5 mg/kg, 75 mg/kg or 150 mg/kg twice daily (BID) for 28 days, Human Treg suppression assay with HEC (1% hydroxyethyl cellulose (HEC) in 25 mM CD4 cells purified from heathy donor blood (New York phosphate buffer, pH = 2) as control vehicle. For + 6 5 Blood Center, NYC) using the Rosetta CD4 T cell combination therapy studies, 1 × 10 CT26 or 5 × 10 enrichment kit (Stem Cell Technologies). CD25 and MC38 cells were injected subcutaneously into the flank − + CD25 T cells were then isolated using human CD25 T of Balb/c or C57BL/6 mice, respectively. Galunisertib cell microbeads (Miltenyi). Naïve CD25 T cells were was dosed at 75 mg/kg BID for 21 days and anti-PD-L1 labeled with 1 mM CFSE (Invitrogen) as described antibody (clone 178G7; Lilly NYC) or Rat IgG antibody − + above. CD25 naïve T cells and CD25 Tregs were re- was given 3 times intraperitoneal at 500μg/dose every suspended in complete RPMI media (Gibco) and plated 7 days (q7dx3). For depletion of CD8 T cells, mice onto 96-well plates at indicated ratios of Treg cells to were injected i.p. with 200 μg of CD8a antibody naïve T cells with 5 × 10 cells/well in total; except for (clone 53–6.7; eBioscience) on day 1, 2 and 3 after Tregs alone and untreated naïve T cells which were tumor challenge, followed by injection of 200 μg plated at 2.5 × 10 cells/well. Cells were then stimulated weekly throughout the experiment. For all studies, with CD3/CD28/CD2 antibody coated beads (Miltenyi) mice were randomized by body weight or tumor vol- at a bead to cell ratio of 1:1with unstimulated CD25 ume into groups of 8–15 mice prior to treatment. For naïve T cells as a control. Galunisertib (0.1μMto MOA experiments, separate subgroups of 3–5ani- 10 μM) was added with DMSO as vehicle control. mals/MOA timepoint were pre-assigned at study initi- Proliferation was measured by CFSE dilution as above ation and not included in survival evaluation. Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 4 of 15 Tumor volume was calculated using a formula: Tumor background MFI of blank well; “Adjusted Net MFI”, cal- 3 2 Volume (mm )= π/6 * Length * Width . Animals were culated: if MFI > lower limit of detection (LLOD, back- sacrificed due to progressive disease if tumor burden ground MFI + 3 standard deviations), then “Adjusted was greater than 2500 mm and if growth would surpass Net MFI” = “Net MFI”, if MFI < LLOD, then “Adjusted 2500 mm before the next scheduled measurement. For Net MFI” = LLOD – background. Adjusted Net MFI was rechallenge experiments mice with complete regressions used to calculate relative gene expression normalizing (tumor volume < 14 mm ) were rechallenged as each gene to the geometric mean of the MFI of selected indicated and followed for ~ 30 days. housekeeping genes (HKG) (adjusted net MFI/geometric Tumor volumes compared to control (%T/C) were mean HKG MFI) multiplied by a scaling factor of 100. calculated as %T/C = 100 x ΔT/ΔC, whereby ΔT= Data visualizations were done using TIBCO Spotfire® mean tumor volume of treated group, and ΔC = mean software (Spotfire, Somerville, MA). tumor volume of the control (vehicle) on indicated dayminus themean tumor volumeonthe baseline. Results Statistical analysis was performed by two-way re- Galunisertib blocks TGFβ1 mediated suppression of naïve peated measures analysis using the log transformation T cell proliferation and blocks Treg mediated suppression of tumor volume. Predefined pairwise comparisons of naïve T cells were conducted as indicated. TGFβ signaling plays an important role in suppressing an immune reaction and inducing tolerance. In particu- Isolation of tumor-infiltrating cells and lymphoid tissue lar, TGFβ signaling inhibits innate and adaptive immune cells functions and induces suppressive immune cells. To test Tumors and spleens were harvested from individual if galunisertib could rescue TGFβ suppressed immune mice at specific MOA time points after tumor cell in- cell subsets, naïve T cell suppression assays were estab- oculation. Single cell suspensions were made by hom- lished, and suppression mediated by TGFβ1orbyT ogenizing each tissue separately through 40 μm nylon regulatory cells (Tregs) was tested in in vitro culture sys- mesh strainers into complete media (RPMI+ 10% FBS). tems. For these experiments, naïve human CD8 T cells After RBC lysis (ACK lysis buffer; Gibco) when required, were stimulated with anti-CD3/anti-CD28 beads in the all samples were washed and re-suspended in FACS buf- presence or absence of TGFβ1. As shown in Fig. 1a, fer (PBS + 4% BSA) for fresh FACS analysis or snap fro- while TGFβ1 potently suppressed the proliferation of zen for gene expression analysis. CD8 T cells, addition of galunisertib resulted in a dose- dependent rescue of proliferation in the TGFβ1treated FACS analysis cultures, with enhanced proliferation observed at the Single cell suspensions prepared from mouse tumors higher doses of galunisertib. To evaluate the ability of and spleens were pre-incubated with 1 μl/ml anti-CD16/ galunisertib to modulate Treg suppressive activity, + + 32 monoclonal antibody (Fc block; Tonbo) for 30 min at CD4 CD25 Treg cells were co-cultured with naïve T cells + − 4 °C and then stained with indicated fluorochrome- (CD4 CD25 ) in the presence of anti-CD3/anti-28/anti- + + conjugated antibodies (eBioscience) and a fixable CD2 stimulation. While CD4 CD25 Tregs potently viability dye (Life Technologies). Labeled cells were suppressed naïve T cell proliferation, addition of acquired BD LSRFortessa and data processed using galunisertib fully reversed the suppression of proliferation, FlowJo software (Treestar). demonstrating a role for galunisertib in reversing Treg mediated immune suppression (Fig. 1b). Quantigene® gene expression analysis Total RNA was isolated from snap frozen tumor tissue Galunisertib monotherapy induces regression of lysed using the MagMax™ 96 Total RNA isolation kit immunogenic 4T1-LP tumors (Life Technologies) homogenized with steel beads on a To explore the impact of galunisertib monotherapy on TissueLyser (Qiagen) for 2 min at 25 Hz. Samples were preventing growth of established tumors, we utilized the processed washed, and incubated with DNase, on the poorly immunogenic murine triple negative breast MagMax™ Express 96 Processor. 500 ng of RNA was tumor model, 4T1, and a variant engineered to express incubated in duplicate with QuantiGene® magnetic cap- luciferase, (4T1-LP). For these experiments, immune ture beads, probesets, and blocking reagent (Affymetrix) competent Balb/c mice were injected orthotopically in and analyzed on the FlexMap 3D® (ThermoFisher, Wal- the mammary fat pad with 4T1-LP or 4T1 tumors. tham, MA). Level of RNA detection was determined by When tumors were well established (~300mm ,~8– mean fluorescence intensity (MFI) and converted into 11 days after implantation), animals were treated with adjusted net MFI using an in-house quality control ana- galunisertib at 75 mg/kg BID. Animals were treated for lysis script. “Net MFI”: sample was calculated as MFI – 28 days then followed for tumor growth. In the 4T1-LP Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 5 of 15 ab ns + TGF + Tregs Fig. 1 Galunisertib blocks TGFβ1 mediated suppression of naïve T cell proliferation and blocks Treg mediated suppression of naïve T cells. a Human CD8 cells purified from healthy donor whole blood where labeled with CFSE and stimulated with anti-CD3/CD28 beads in the presence of TGFβ1 and galunisertib. CD8 T cell proliferation was measured by assessing CFSE dilation by flow cytometry after 5 days of culture. Percent recovery of CD8 T cell proliferation at different concentrations of galunisertib (0-10 μM) as indicated and representative histograms are shown. + + DMSO was used as vehicle control. Data shown are combined data of 4 healthy donors. b Human CD4 CD25 cells purified from healthy donor + − whole blood where labeled with CFSE and stimulated with anti-CD3/CD28 beads in the presence of galunisertib. Autologous CD4 CD25 Treg + − were added at a 1:1 ratio of Treg cells to naïve T cells. Naïve CD4 CD25 T cell proliferation was measured by assessing CFSE dilation by flow + − cytometry after 5–7 days of culture. Percent recovery of CD4 CD25 T cell proliferation at different concentrations of galunisertib (0-10 μM) as indicated and representative histograms are shown. Data shown are combined data of 4 healthy donors; representative of 3 independent experiments. One-way ANOVA with Dunnett’s test was used to compare the galunisertib treatments to the DMSO treatment. ****: p ≤ 0.0001; ***: p ≤ 0.001; **: p ≤ 0.01; *: p ≤ 0.05; ns: p ≥ 0.05 model, the majority of mice (10/12) responded to galuni- eventually progressed without treatment. The spontan- sertib therapy, including 4/12 complete responses (Fig. eous activity we observe in a few of the 4T1-LP control 2a); in contrast, none of the poorly-immunogenic 4T1 mice (Fig. 2a) is likely reflective of an immune response bearing mice responded to galunisertib therapy (Fig. 2b), to the implanted tumors, and this immune response suggesting that the presence of a foreign antigen (i.e. LP) may in fact be the mechanism by which galunisertib is , potentially enhanced the ability of galunisertib to in- so much more active as a monotherapy in 4T1-LP com- duce the rejection of the 4T1-LP derivative. In a previ- pared to 4T1 parental. We speculate that the immuno- ous study, a survival benefit advantage with galunisertib genic nature of 4T1-LP is likely what impacts was observed in the poorly-immunogenic 4T1 tumor galunisertib monotherapy activity in this model, while in model (Yingling et al., [25]). This may reflect the early less immunogenic tumor models, combination with start of treatment in that study (day 4 after tumor im- anti-PD-L1 is needed (described below). plantation compared to 8–11 days in the study pre- To further evaluate and interrogate the impact of sented here) or it may be a result of an anti-metastatic galunisertib on anti-tumor activity, mice bearing 4T1- activity rather than an effect only on primary tumor LP tumors in the mammary fat pad were treated for growth. 28 days (starting at day 8 post tumor challenge) with A few mice in the vehicle control group in the 4T1-LP three different doses of galunisertib (37.5, 75 and model, but not in the parental 4T1 model, showed an 150 mg/kg BID). Anti-tumor activity was observed at initial tumor response before eventually progressing (Fig. all 3 doses of galunisertib, with a dose dependent in- 2a), suggesting that spontaneous responses to immuno- crease in activity as assessed by both mean tumor genic tumor cell lines can occur in some mice. This may volume and CR (Fig. 3a-d). Anti-tumor activity was reflect the different T cell repertoire between individual observed following an initial growth pattern similar to mice as we did not use TCR transgenic mice, or suggest- controls, indicating a delayed response to treatment ing that these mice developed an immune response to a and possible immune mediated mechanism. Following dominant CTL epitope of LP, which may lead to reduced cessation of therapy on day 36 (28 days of treatment), tumor invasiveness and spontaneous regression. responding mice progressed to complete responders [28]. Although, a few mice of the control group in a dose-dependent manner, with 1/10 CR in mice showed initial spontaneous activity, all untreated tumors treated with 37.5 mg/kg BID, 3/10 CRs in mice % of Max Proliferion Compared Control (+/- SEM) Control DMSO Gal 0.1µM Gal 0.3µM Gal 1µM Gal 3µM Gal 10µM % Rescue Naive T cell Proliferation Naive T cells DMSO Gal 1.1µM Gal 3.3µM Gal 10µM Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 6 of 15 Importantly, PK/PD profiling studies of galunisertib 4T1-LP Model suggest that administration of 75 mg/kg BID in preclin- Individual Tumor Growth Curves ical models or 150 mg/kg BID in patients can achieve significant target modulation in vivo over a 24-h period [1, 29]. Thus, we show anti-tumor activity with galuni- sertib at clinically relevant doses. To begin to understand how galunisertib treatment modulated immune cells within the tumor, tumors from mice galunisertib treated or vehicle control mice were harvested 8 days after therapy initiation and the changes in T cell infiltration were analyzed by flow cytometry. Control Relative to control animals, a modest increase in both Galunisertib CD3 T cells, mainly CD8 T cells, and NK cells was observed in tumors of mice treated with the clinically 020 40 60 80 relevant dose of 75 mg/kg galunisertib (Fig. 3c), indicat- Gal (BID): ing a role of galunisertib on T-cell expansion or T-cell Day Post Tumor Challenge trafficking to the tumor site. These differences did not reach statistical significance. No significant changes were 4T1 Parental Model observed in the myeloid compartment in tumors of galu- Individual Tumor Growth Curves nisertib treated mice compared to control treated mice in this model. However, only the number of myeloid cells was analyzed and not the function; thus, whether galunisertib induces reprogramming toward an antitu- mor phenotype was not explored. This may also reflect the time point of tumor collection. To this end, a prior study with anti-mouse TGFβRII showed modulation of 500 MDSCs by blocking the TGFβ signaling pathway [13]. Control Galunisertib Galunisertib monotherapy induces immunologic memory 100 and demonstrates antigen spreading 4T1-LP tumor bearing mice that completely responded 020 40 60 80 to galunisertib therapy remained tumor free for up to Gal (BID): 85 days (49 days after treatment completed) (Fig. 3a), Day Post Tumor Challenge indicating a durable response. To test the ability of galu- Fig. 2 Galunisertib monotherapy induces regression of nisertib to induce immunologic memory to 4T1-LP tu- immunogenic 4T1-LP variant breast tumors. Mean and individual mors, mice which had completely regressed 4T1-LP tumor growth curves for Balb/c mice injected orthotopically in the mammary fat pad with 4T1-LP (a) or parental 4T1 (b) tumor cells were re-challenged orthotopically with 4T1-LP on the and treated with galunisertib (75 mg/kg BID) when tumors reached opposite flank of the original tumor injection site and ~300mm (6–8 days after implantation). The number of mice/group additionally received a primary challenge of a different rejecting tumors (complete responders, CRs) was: control (0/10 mice) triple negative breast cancer tumor, EMT6-LM2, on the and galunisertib (4/12) for 4T1-LP, and control (0/10 mice) and flank of the original tumor injection site. In all mice galunisertib (0/12) for 4T1 parental as indicated. Data shown are representative of two independent experiments with tested, complete responders rejected the re-challenge 10–12 mice/group with the 4T1-LP tumors (Fig. 3d, left panel), but did not reject EMT6-LM2 tumors (Fig. 3d, middle panel), dem- treated with 75 mg/kg BID, and 5/10 CRs observed in onstrating immunologic memory to the 4T1-LP tumor mice treated with 150 mg/kg BID (Fig. 3a). CR mice cells, but not the heterologous tumor. To evaluate the remained tumor free for an additional 49 days in the potential for epitope spreading as a result of galunisertib absence of further treatment. These data indicate that anti-tumor activity, mice which had completely galunisertib induces a potent, dose-dependent durable regressed 4T1-LP after being treated with 75 mg/kg of anti-tumor response. Metastases to lungs were not galunisertib were re-challenged in a separate experiment observed in this tumor model. with the poorly immunogenic parental 4T1 tumors, Treatments with galunisertib were well-tolerated which lack the immunogenic LP transgene and is not re- with no body weight loss observed with any of the sponsive to de-novo galunisertib monotherapy (Fig. 2b); doses tested (Fig. 3b). in all mice tested, 4T1-LP complete repressors also Tumor Volume mm Tumor Volume mm Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 7 of 15 Mean 4T1-LP Tumor Volume Galunisertib Treatment Control 37.5 mg/kg 75 mg/kg 150 mg/kg 0 1020 304050 60 Gal (BID): Days Post Tumor Challenge Galunisertib 37.5mg/kg Galunisertib 75mg/kg Galunisertib 150mg/kg 2500 2500 2500 Control Control Control 37.5mg/kg 75mg/kg 150mg/kg 2000 2000 2000 1500 1500 1500 1000 1000 1000 500 500 500 0 0 0 0 10203040 50607080 0 10203040 50607080 0 10203040 50607080 Gal (BID): Gal (BID): Gal (BID): Days Post Tumor Challenge Days Post Tumor Challenge Days Post Tumor Challenge b Mean Body Weight d Galunisertib Treatment 4T1-LP Rechallenge EMT6-LM2 Challenge 30 5000 5000 Control Galunisertib 37.5 mg/kg Galunisertib 37.5 mg/kg 37.5 mg/kg Galunisertib 75 mg/kg Galunisertib 75 mg/kg 75 mg/kg Galunisertib 150 mg/kg Galunisertib 150 mg/kg 150 mg/kg 3000 3000 10 0 0 10 203040 5060 7080 82 85 88 91 94 97 100 103 106 109 82 85 88 91 94 97 100 103 106 109 Gal (BID): Days Post Primary Tumor Challenge Days Post Primary Tumor Challenge Days Post Tumor Challenge Galunisertib (mg/kg): CD45 intra-tumor c e Parental 4T1 Rechallenge Parental 4T1 0 37.5 75 150 Individual Tumor Growth Naive Mice 2000 2000 0.88% 3.37% 1.27% 3.14% 0.81% 3.05% 0.51% 1.69% 400 400 NK Cells (DX5) 0 0 85 90 95 100 105 110 85 90 95 100 105 110 Day Post Tumor Challenge Day Post Tumor Challenge Fig. 3 (See legend on next page.) T cells (CD3) Mean Tumor Volume mm (+/-SEM) Tumor Volume mm Mean body weight (g) (+/- SEM) Tumor Volume mm 3 3 Tumor Volume mm Mean Tumor Volume mm (+/-SEM) Rechallenge Tumor Volume mm 3 3 Tumor Volume mm Mean Tumor Volume mm (+/-SEM) Rechallenge Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 8 of 15 (See figure on previous page.) Fig. 3 Galunisertib monotherapy displays dose dependent activity against established orthotopic 4T1-LP tumors with immunologic memory and antigen spreading. a Mean and individual tumor growth curves for Balb/c mice injected orthotopically in the mammary fat pad with 4T1-LP tumor cells and treated with galunisertib (37.5, 75 or 150 mg/kg BID) when tumors reached ~ 300 mm3 (8 days after implantation). The number of CRs were: 1/10, 3/10 and 5/10 for mice treated with Galunisertib at 37.5 mg/kg, 75 mg/kg and 150 mg/kg, respectively. b Percent body weight change on average of mice treated with galunisertib at 37.5 mg/kg, 75 mg/kg and 150 mg/kg. c Frequencies and representative plots of CD3 and NK T cells of total live CD45 cells in in single cell suspensions prepared from tumors harvested after 8 days of galunisertib treatment. Tumors from 5 mice/group treated with 75 mg/kg galunisertib or vehicle control were analyzed. Student’s t-tests were used to evaluate statistical significance (ns: p ≥ 0.05). d Mice which had regressed 4T1-LP tumors after treatment with galunisertib (37.5, 75 or 150 mg/kg BID for 28 days; as shown in a) were rechallenged orthotopically with 4T1-LP in one mammary fat pad and received a primary challenge of a different triple negative breast cancer tumor, EMT6-LM2, in an alternate fat pad, at day 85 post the original 4T1-LP tumor challenge. Average tumor growth curves of secondary 4T1-LP and EMT-6-LM2 challenge are shown for each group. e Mice which had regressed 4T1-LP tumors after treatment with galunisertib (75 mg/kg BID for 28 days) were rechallenged orthotopically with the poorly immunogenic 4T1 parental tumor cells at day 85 post the original 4T1-LP tumor challenge. Average tumor growth curves of secondary 4T1-LP challenge are shown. Individual tumor growth curves of naïve mice injected orthotopically with 4T1 parental tumors are shown as a control. Data shown are representative of two independent experiments with 10–12 mice/group rejected the parental 4T1 challenge (Fig. 3e, right panel), tumors indicating a requirement for an adaptive immune demonstrating the potential for galunisertib anti-tumor system, and in particular CD8 T cells, in this model activity to mediate antigen spreading. (Fig. 4). Galunisertib anti-tumor activity in the 4T1-LP model is Combined blockade of TGFβR1 and PD-L1 enhnaces CD8 T cell dependent regression of tumors The delayed response to galunisertib and the modest in- Because the in vitro and in vivo galunisertib monother- crease in tumor-infiltrating lymphocytes (TILs) in apy data strongly suggested that galunisertib was able to treated mice suggested that the adaptive immune re- modulate anti-tumor T cell immunity, we investigated sponse may be involved in the mechanism of tumor re- whether galunisertib could synergize with PD-L1 check- jection following galunisertib therapy. To evaluate the point blockade and result in improved tumor regres- role of the adaptive immune response in galunisertib sions. Anaphylactic reactions have been reported with anti-tumor activity, studies using the orthotopic 4T1-LP PD-L1 and PD1 monoclonal antibodies in the ortho- −/− model were carried out in the RAG mice or in Balb/c tropic 4T1 tumor model [30] (Mall et al., [31]), thus, for mice depleted of CD8 T cells by treatment with an these studies we utilized the CT26 mouse model well −/− immune depleting anti-CD8α antibody. In both RAG known to be responsive to various degrees to PD-1 axis and CD8 T cell depleted mice bearing 4T1-LP tumors, immunotherapy [32]. Balb/c mice were injected with galunisertib therapy was unable to induce regression of CT26 tumor cells and treated with galunisertib, anti-PD- ab RAG-/- Mice CD8α Depleted Mice Control Control Galunisertib 75mg/kg Control CD8α depleted 1500 Galunisertib 75mg/kg Galunisertib CD8α depleted 0 5 10 15 20 0 5 10 15 20 Gal (BID): Gal (BID): Days Post Tumor Challenge Days Post Tumor Challenge −/− Fig. 4 Galunisertib anti-tumor activity in the 4T1-LP model is CD8 T cell dependent. a Mean tumor growth curves for Rag mice injected orthotopically in the mammary fat pad with 4T1-LP tumor cells and treated with galunisertib (75 mg/kg BID × 28 days) when tumors reached ~ 300 mm3 (8 days after implantation). b Mean tumor growth curves for Balb/c mice injected orthotopically in the mammary fat pad with 4 T1-LP tumor cells and treated with galunisertib (75 mg/kg BID × 28 days) plus depleting antibody for CD8α (200 μg/mouse). Treatment was initiated when tumors reached ~ 300 mm3 (8 days after implantation). CD8α monoclonal antibody was injected on day 1, 2 and 3 after tumor challenge, followed by injection weekly throughout the experiment. Data represent two independent experiments with 8 mice/group Mean Tumor Volume mm (+/-SEM) Mean Tumor Volume mm (+/-SEM) Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 9 of 15 L1 or a combination of both for 21 days. Treatment was active in this setting with 5/15 and 3/14 CR respectively initiated on Day 6 when tumors were ~100mm and while the combination of anti-PD-L1 and galunisertib continued for 28 days. The mean tumor growth under therapy resulted in marked enhancement of response (9/ each condition is presented in Fig. 5 (left panel), and 14 CRs) and all animals responding to treatment (Fig. 5). shows that while both galunsertib and anti-PD-L1 ther- There was a significant antitumor benefit with the com- apy were active in this model, the combination of galuni- bination group versus each monotherapy (p < .001). To sertib and anti-PD-L1 resulted in enhanced anti-tumor test if each treatment could result in immunologic mem- activity. As shown in the individual animal plots, anti- ory, mice with CRs were re-challenged with CT26 PD-L1 or galunisertib monotherapy were modestly tumors 85 days after primary tumor challenge (51 days Mean CT26 Tumor Volume Galunisertib and Anti-PD-L1 Therapy Rat IgG Anti-PD-L1 Galunisertib Combo 0 5 10 15 20 25 Gal (BID): Anti-PD-L1: Days Post Tumor Challenge Galunisertib Monotherapy Galunisertib and Anti-PD-L1 Combination Therapy Anti-PD-L1 Monotherapy 3000 3000 Rat IgG Rat IgG Rat IgG Galunisertib Combo PD-L1 2500 2500 2500 2000 2000 1500 1500 1000 1000 500 500 0 0 0 102030405060 0 102030405060 0 1020 3040 506070 8090 100 Gal (BID): Anti-PD-L1: Gal (BID): Anti-PD-L1: Days Post Tumor Challenge Days Post Tumor Challenge Days Post Tumor Challenge b c Mean MC38 Tumor Volume Secondary CT26 Rechallenge Galunisertib and Anti-PD-L1 Therapy Rat IgG Galunisertib * Anti-PD-L1 Anti-PD-L1 Galunisertib Combo Combo 80 85 90 95 100 105 0 5 10 15 20 Days Post Primary Tumor Challenge Gal (BID): Anti-PD-L1: Day Post Tumor Challenge Fig. 5 Combined blockade of TGFβR1 with Galunisertib and PD-L1 induce robust regression of murine colon tumors. Mean and individual tumor growth curves for Balb/c mice injected with CT26 tumor cells and treated with galunisertib (75 mg/kg BID for 28 days) and/or anti-PD-L1 antibody (500 μg/dose, q7dx3) when tumors reached ~100mm (6 days after implantation). The percentages of CRs were: control (0/15 mice), galunisertib monotherapy (3/14), anti-PD-L1 monotherapy (5/15) and combination therapy (9/14). b Mice which had regressed CT26 tumors after treatment with galunisertib and/or anti-PD-L1 were rechallenged with CT26 tumor cells on the contralateral flank at day 85 post the original tumor challenge. Individual tumor growth curves of secondary CT26 challenge are shown for each treatment group. c Mean and individual tumor growth curves for C57BL/6 mice injected with MC38 tumor cells and treated with galunisertib (75 mg/kg BID for 21 days) and/or anti-PD-L1 antibody (500 μg/dose, q7dx3). Treatment was initiated 3 days after tumor cell implantation. Data shown are representative of two independent experiments with 10–12 mice/group 3 3 Tumor Volume mm Mean Tumor Volume mm (+/-SEM) Tumor Volume mm Rechallenge Tumor Volume mm Mean Tumor Volume mm (+/-SEM) Tumor Volume mm Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 10 of 15 after initial treatment cessation). All complete re- mice were collected day 10, 16, and 22 after tumor chal- sponders in monotherapy and combination treatment lenge (i.e. 4, 10, and 16 days after initiation of therapy) groups rejected the re-challenge with the CT26 tumors and subjected to high-content molecular profiling using TM (Fig. 5b, left panel); however, one animal from the com- a custom designed Quantigene gene panel to detect T bination cohort, defined as a long-term partial responder cell activation and intra-tumoral inflammation (Table 1). (indicated by * in Fig. 5a right panel) was unable to reject Galunisertib monotherapy (75 mg/kg) did not appre- secondary tumor challenge. The ability of galunisertib to ciably alter the set of immune genes analyzed relative to enhance the activity of anti-PD-L1 immunotherapy was control tumors at any time point evaluated (Fig. 6, top confirmed in the MC38 tumor model, which is historically panel). Anti-PD-L1 monotherapy resulted in an en- less responsive to checkpoint immunotherapy and consid- hanced T cell infiltration and activation profile, exempli- ered to be more myeloid biology driven [32]. In this fied by the increase in multiple immune activation and model, where treatment began on day 3 after tumor chal- inflammation transcripts such as Ccl5, Itgax, Icam1, lenge, similar monotherapy and combination therapy ac- Foxp3, Lag3 by day 22 (Fig. 6a). On the other hand, the tivity was observed albeit with more moderate activity combination treatment demonstrated an early signature overall (Fig. 5c). of enhanced T cell activation and inflammation exempli- fied by the upregulation of transcripts for Ifnγ, Lag3, Combination of galunisertib and anti-PD-L1 checkpoint Ccl3, Ccl4, Ccl5, and Tnfrsf18) beginning on Day 16 blockade induces an intra-tumor immune related gene (after only 10 days of therapy, where only a minor expression profile that is accelerated and enhanced change in activation was detected with anti-PD-L1 compared to anti-PD-L1 monotherapy monotherapy), and continuing at day 22 with an en- To further elucidate the mechanism of action of the hanced gene expression related to T cell infiltration combination activity of galunisertib and anti-PD-L1, (Ptprc, Cd8b1, Cd3e, and Cd4) and T cell activation and gene expression studies were carried out on tumors inflammation (Il2, Il4, Il17a, Lag3, Ifnγ, Ifnα, Ifnβ1, from CT26 tumor bearing mice treated with control, Foxp3, Cd274, and Pdcdlg2) (Fig. 6a). The gene profile anti-PD-L1, galunisertib or a combination of anti-PD-L1 for the combination cohort was similar but more robust plus galunisertib. For these studies, tumors from treated compared to PD-L1 monotherapy detected at the later Table 1 Custom designed Quantigene™ gene panel to detect T cell activation and intra-tumoral inflammation Cell type-specific Coinhibitory & Cytokines & Immunosuppressive Markers of Angiogenesis; EMT TGFb HKGs markers Costimulatory Chemokines Enzymes T cell activation Endothelial activation markers pathway Cd14 4632428N05Rik (Vista) Ccl2 (MCP-1) Arg1 Cd69 Cdh5 Cdh1 Smad4 Gus Cd3e Cd200r1 Ccl3 (MIP-1a) Ido1 Gzmb Hif1a Epcam Tgfb1 Hprt Cd4 Cd274 (PD-L1) Ccl4 (MIP-1b) Mpo Ifng Vcam1 Fap Tgfb2 Ppib CD68 Cd40lg Ccl5 (RANTES) Nos2 Vegfa Snai1 Tgfb3 Rps18 Cd8b1 Cd86 Csf2 (GM-CSF) Tdo2 Vegfc Twist1 Tgfbr1 Foxp3 Havcr2 (TIM3) Cxcl1 Icam1 (CD54) Vim Tgfbr2 Itgam (CD11B) Icos Ifna2 Sele Cspg4 Itgax (CD11C) Lag3 Ifnb1 Klrk1 Pdcd1 (PD-1) Il10 (CSIF) Ms4a1 Pdcd1lg2 (PD-L2) Il13 Ptprc (CD45) Pvr Il17a Pvrl2 Il2 Tigit Il4 Timd4 (TIM4) Il5 Tnfrsf18 (GITR) Il6 Tnfrsf4 (OX40) Il9 Tnfrsf9 (4-1BB) Tnf Tnfsf18 (GITRL) Tnfsf4 Tnfsf9 (CD137L, 4-1BBL) Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 11 of 15 Fig. 6 Combination of galunisertib and anti-PD-L1 checkpoint blockade induces an intra-tumor immune related gene expression profile that is accelerated and enhanced compared to anti-PD-L1 monotherapy. Log2 Fold Change in gene expression of galunisertib (75 mg/kg BID for 28 days) and/or anti-PD-L1 (500 μg/dose, q7dx3) treated CT26 tumors against vehicle control. Differential expression and p-value was evaluated by two-way ANOVA testing. Labelled and highlighted genes are log2FC > 1, p-value< 0.05, are shown in Volcano Plots (a) and Venn Diagrams (b). Tumors were harvested and analyzed at different time points after tumor challenge: day 10, 16 and 22 (treatments were initiated at 6 days after implantation) time point (day 22) (Fig. 6a and b). Interestingly, the treat- during therapy, and T cell frequencies were similar for ments also resulted in increased expression of some im- monotherapy PD-L1 and combination therapy (data not munosuppressive genes, such as Ido1, Mpo, Nos2 and shown), suggesting that the effects of combination were Tdo2, which may reflect a counter-regulatory mechanism modulated at the effector function level. Finally, we ob- induced by the tumor and/or myeloid cells in response to served that some genes such as FAP were upregulated at enhanced IFNγ production by anti-PD-L1 or combination day 22 upon anti-PD-L1 treatment but not with combin- treated tumor-infiltrating T cells. In support of this, pre- ation therapy, suggesting that galunisertib may be acting clinical work using murine tumor models have shown that by remodeling tumor stroma (Fig. 6a), as previously de- dual targeting of IDO and checkpoints results in enhanced scribed in the literature with other inhibitors of the TGFβ anti-tumor immunity [33–35]. Overall, combination ther- pathway [13, 36]. apy resulted in an accelerated and more robust increase in genes indicative of T cell activation compared to either Discussion monotherapy suggesting that inhibiting immune suppres- TGFβ plays diverse and important roles in tumorigen- sion with galunisertib may accelerate the biological activity esis, including tumor proliferation, invasion and metas- of anti-PD-L1. Examination of immune cell subset fre- tasis, inflammation, angiogenesis, and escape of immune quencies in tumors by flow did not detect major changes surveillance. Therefore, targeting the TGFβ signaling Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 12 of 15 pathway has been an attractive objective for cancer ther- tumor activity in immune-competent murine tumor apy, and several drugs have been identified and are models. We show that galunisertib monotherapy induces under clinical development [37–39]. Galunisertib is a dose-dependent regression of well-established immuno- small molecule inhibitor of the kinase domain of genic 4T1-LP breast tumors. The responses were dur- TGFβRI. Binding of TGFβ ligands to TGFβRII is the first able with immunological memory as demonstrated by step to initiate activation of the TGFβ signaling pathway. rechallenge experiments with 4T1-LP tumors as well as Once bound to TGFβRII, this ligand/receptor complex a second triple negative breast tumor cell line. Of note, binds to TGFβRI to form a heterotrimeric complex. For- mice that rejected the immunogenic 4T1-LP tumors mation of this complex results in phosphorylation of the were also able to reject 4T1 parental cells upon rechal- serine/threonine kinase domains of the receptors, lenge, suggesting the development of a secondary followed by activation of the canonical SMAD2/3 signal- immune response via antigen spreading as a conse- ing pathways as well as non-canonical (i.e. MAPK) path- quence of effective tumor targeting. The anti-tumor ways [39]. These pathways modulate transcription of activity of galunisertib in the 4T1-LP tumor model was numerous target genes, resulting in a variety of effects. CD8 T cell dependent and associated with a modest By blocking the kinase domain of TGFβRI, galunisertib increase in T-cell infiltration in tumors. The increase may effectively inhibit signaling via the TGFβ pathway. was modest and did not reach statistical significance Preclinical and clinical research on galunisertib, though, which might reflect the time point chosen for including the treatment of over 800 patients, has dem- tumor collection. It is well established in the immuno- onstrated that SMIs of TGFβ can safely be developed for oncology field that the spatial distribution and location of clinical testing, provided there is an adequate under- immune cells is highly important. In fact, the recent publi- standing of the pharmacokinetic/pharmacodynamics cations combining TGFβ inhibition and PD-L1 blockade (PK/PD) relationship, as most of the toxicities in animal show that the main mechanism of action of TGFβ inhib- models that were of concern prior to the start of clinical ition is to increase T-cell infiltration into tumor [26, 27]. development of galunisertib have not been observed in In addition, using an anti-TGFβRII blocking antibody we humans [1]. Furthermore, preclinical and clinical efforts have previously shown that blocking TGFβ signaling in suggest that the biology of the TGFβ inhibition is largely the EMT6 tumor model induces immune infiltration [13]. dependent on the microenvironment, perhaps more than We did not investigate metastasis to lungs in either originally anticipated. For example, TGFβ1 is a potent the 4T1 or 4T1-LP tumor models used here. However, inducer of angiogenesis [40], by directly inducing VEGF we have previously shown that the anti-mouse TGFβRII expression [41], or recruiting other cells, such as mono- antibody significantly inhibits the growth of established cytes, which in turn secrete proangiogenic molecules 4T1-parental primary tumors and diminishes the spon- [42]. TGFβ can also manipulate the tumor microenvir- taneous pulmonary metastasis [13]. In addition, it was onment to favor the evasion of cancer cells from im- shown that galunisertib in combination with anti- mune surveillance via tampering with the antitumor CTLA4 therapy suppresses both primary melanoma functions of T cells, NK cells, B cells, and other cells tumor growth as well as metastases in a physiological [43–45]. This activity of TGFβ may be mediated through relevant trangenic melanoma model (Hanks et al. [50]). its direct effect on these cells, as well as via its ability to Furthermore, we described that galunisertib inhibits induce Foxp3 Tregs [46]. Both cancer-intrinsic and TGFβ mediated migration of U87MG glioblastoma cells immune-mediated effect of TGFβ in breast cancers have in vitro in a dose-dependent manner [51]. Notably, in been described [47–49]. Thus, a focus on direct tumor this model system, galunisertib reduced baseline migra- cell cytotoxicity may be misleading and provide incon- tion of U87MG cells in the absence of exogenous clusive observations that will not be helpful to advance TGFβ1, presumably by inhibiting autocrine signaling clinical development of future TGFβ inhibitors. Early through TGFβRI. Together this suggests that galuniser- studies using immune-compromised animals may there- tib has the capacity to suppress the development of me- fore also have limited the screening for TGFβ inhibi- tastasis and that TGFβ pathway blockade of the parental tors. It now appears that an active immune response 4T1 model is sufficient to inhibit metastasis to lung. is essential to assess the effect of TGFβ signaling in- Importantly, we have previously shown that the TGFβ hibition in animal models; thus, immune-competent pathway is abrogated upon treatment with galunisertib animal models maybemorepredictivetoevaluate both in vitro and in vivo [51]. We demonstrated that TGFβ inhibitors. Consequently, more novel preclinical galunisertib inhibited TGFβ-induced pSMAD in various testing assays are required than those traditionally tumor cell lines, including 4 T1-LP, in vitro in a dose- used in oncology research. dependent manner [51]. Furthermore, we reported a Here we describe the impact of galunisertib to modu- galunisertib time and dose-dependent inhibition of late the immune system and its ability to enhance anti- endogenous TGFβ-dependent signal transduction in vivo Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 13 of 15 in EMT6-LM2 murine syngenic tumor models [51]. gene panel tested, the dose chosen or the day of collection. These data suggest that the effects of galunisertib are Similar combination therapy activity was observed in on-target. Potential off-target effects of galunisertib are the PD-L1 insensitive tumor model, MC38, albeit with further diminished, as treatment with an anti-mouse more moderate activity overall, suggesting at least TGFβRII antibody similarly inhibits the growth of estab- additive activity with potential for synergy when tar- lished mouse 4T1 and EMT6 primary [13]. geting the TGFβ and PD-1 pathways. The anti-tumor Immunotherapeutic strategies such as immune check- activity of galunisertib was tested in a broad range of point blockade have shown significant promise for treat- murine tumor models with similar results, further ment of cancers resistant to conventional modalities, suggesting that TGFβ inhibition is immune mediated leading to Food and Drug Administration (FDA) and thus not restricted to specific tumor indications. approval in advanced melanoma, renal cell carcinoma Finally, we show that galunisertib reverses both TGFβ and non-small cell lung cancer (NSCLC) [52]. Despite and Treg mediated suppression of T cell proliferation in clinical results, even with combined checkpoint blockade human cell cultures in vitro, which further highlight the [53], therapeutic success has so far been limited to a important role of galunisertib to overcome immune sup- subset of patients, calling for identification of markers pression and promote anti-tumor immunity. predicting response, identification of resistance mecha- Taken together, the results presented here demonstrate nisms and development of combinatorial therapeutic the impact of blocking TGFβ signaling and provide a approaches. To this end, TGFβ pathway inhibition repre- strong incentive to clinically explore the potential of sents an attractive strategy with its multitude of effects galunisertib treatment to enhance the development of on cancer progression and on the immune system to en- anti-tumor T cell immunity, which may be enhanced by hance the development of anti-tumor T-cell immunity. combinations with immune checkpoint inhibitors. Our Indeed, a recent study by Powles et al., reports that lack results expand on other reports demonstrating that sys- of response to atzeolizumab (anti-PDL1) in bladder can- temic treatment with monoclonal antibodies targeting cer patients was associated with an immune-excluded the TGFβ ligands or the TGFβRII inhibit metastatic phenotype that corresponded with active TGFβ in peritu- invasion of breast cancer cells in murine tumor models moral stroma and a signature of TGFβ signaling [26]. Using [2, 13], and previous work reporting that blocking TGFβ mouse models that recapitulate the immune-excluded signaling with SMIs suppresses metastasis in murine phenotype they further show that co-administration of pancreatic cancer models [54], and enhances radiation blocking antibodies to TGFβ and PDL1 reduced TGFβ sig- response and prolongs survival in xenograft models of naling, facilitated T-cell penetration of tumors, and pro- glioblastoma [55]. voked vigorous anti-tumor immunity leading to tumor Galunisertib continues to advance in clinical trials hav- regressions. In a second recent study published by Batlle ing completed Phase I [56] and is currently under inves- and colleagues, combinatorial activity of galunisertib with tigation in several Phase I and Phase II trials. Thus far, anti-PDL1 in murine colon cancer models was recently de- galunisertib has been very well-tolerated as a first-in- scribed [27]. Combination therapy induced pronounced im- class, oral cancer therapy, and remains a promising com- mune responses which eradicated most metastases, pound in clinical development (http://clinicaltrials.gov/ prolonged recurrence-free survival, and was associated with ct2/results?term=LY2157299). Our data presented here disruption of a T-cell exclusion phenotype. These results support continued clinical development of galunisertib suggest that clinical co-administration of TGFβ and PDL1 to target tumors dependent on TGFβ-driven biology for blocking agents may provide a subset of patients more growth, metastasis, and immune evasion. Whether TGFβ favorable outcomes; however, preclinical validation was per- inhibition applies to all tumors is not clear at this time. formed with either a research-grade reagent [26]or a sig- For clinical development, patient selection tools, defining nificantly excessive amount of galunisertib (800 mg/kg BID who will most likely benefit from TGFβ inhibition, remain compared to the clinically relevant dose of 75 mg/kg a challenging question. Among others, the activity of described in [1, 29]. In agreement, we demonstrate that TGFβ inhibition appears to be dependent on immune combination of galunisertib with PD-L1 checkpoint block- function; thus, it will be important to investigate new bio- ade results in a robust regression of CT26 tumors when markers that are related to immune responses which may compared to single agents. The observed antitumor benefit help with patient selection in future studies. was associated with enhanced expression of genes indica- tive of immune activation and this gene expression profile Conclusion was accelerated compared to anti-PD-L1 monotherapy. In many advanced cancers, TGFβ ligands are overex- Galunisertib alone resulted in no alteration of any of the pressed and the outcome of signaling is diverted toward tested genes. Considering the critical role of TGFβ in can- disease progression. A concerted effort has therefore cer immunity, we speculate that this may be a result of the been to develop drugs that block TGFβ signaling for Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 14 of 15 therapeutic benefit. Galunisertib is a pharmacological 8. Ganapathy V, et al. Targeting the transforming growth factor-beta pathway inhibits human basal-like breast cancer metastasis. Mol Cancer. 2010;9:122. small molecule inhibitor of the TGFβ pathway that acts 9. 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First-in-human dose study of the novel transforming growth factor-beta receptor I kinase inhibitor LY2157299 monohydrate in patients with advanced cancer and glioma. Clin Cancer Res. 2015;21(3):553–60. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal for ImmunoTherapy of Cancer Springer Journals

Targeting the TGFβ pathway with galunisertib, a TGFβRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade

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Medicine & Public Health; Oncology; Immunology
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Abstract

Background: TGFβ signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFβ’s immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFβ pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses. Results: In vitro treatment with galunisertib reversed TGFβ and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti- tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFβ and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy. Conclusions: Together these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy. Keywords: TGF-β receptor I, Antitumor efficacy, Checkpoint inhibitors, Galunisertib * Correspondence: Driscoll_kyla@lilly.com Rikke B. Holmgaard and David Schaer contributed equally to this work. Eli Lilly and Company, 450 East 29th Street, New York, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 2 of 15 Background progression and anti-tumor immunity. That TGFβ may be Transforming growth factor-beta (TGFβ) has been iden- involved in the maintenance of self-tolerance and patho- tified as a therapeutic target in cancer because of its sig- genesis of systemic inflammatory diseases is indicated in nificant and varied roles to promote tumor growth, studies which show the development of multi-organ in- −/− survival, and metastasis. There are several pharmaco- flammation in Tgfb1 mice [15, 16]. The inflammation −/− logical approaches to block TGFβ signaling, including in Tgfb1 mice is dependent on T cells, which undergo neutralizing antibodies, vaccines, antisense oligonucleo- massive activation [17]. Generation of mice lacking tides and small molecular inhibitors (SMI) [1, 2]. The TGFβRII specifically on T-cells further demonstrates the goal of these therapies is to block the tumor-promoting importance of TGFβ in regulating T-cell responses in vivo, effects of TGFβ, while maintaining its tumor suppressive as mice develop multi-organ inflammation similar to that properties. Emerging data and thought suggest that the seen in TGFβ1−/− mice [12, 18]. efficacy of TGFβ antagonist therapy in cancer might not In addition to the direct effects on effector T cell only derive from direct intrinsic effects on tumor cells, responses, TGFβ can promote immunosuppression via but also involves tumor extrinsic mechanisms acting in direct induction and modulation of regulatory T cells the tumor micro-environment. (Tregs) [19]. TGFβ directly promotes expression of TGFβ plays pleiotropic roles to initiate and progress Foxp3 in CD4 T-cells, converting them to a regulatory cancer including both tumor cell intrinsic and extrinsic phenotype [20]. In addition to induction and mainten- activities. Tumor cell intrinsic activities of the TGFβ ance of Foxp3 expression, TGFβ has also been shown to pathway include autocrine TGFβ driven tumor cell pro- be important in the functional ability of Tregs to sup- liferation and differentiation, epithelial to mesenchymal press immune responses [21, 22], and it has been dem- transition (EMT), invasion and migration, prometastatic onstrated that Tgfb1−/− mice fail to maintain peripheral cytokine production, and autocrine mitogen production Treg cells [21]. TGFβ1-producing myeloid-derived sup- [3, 4]. Tumor cell extrinsic activities include promoting pressor cells (MDSCs) have also been reported at high of increased tumor vascularization, modulation of the levels in the tumor microenvironment [23, 24]. stromal extracellular matrix, induction of and feedback Clinical studies have provided proof of concept data modulation of the hypoxic state and inhibition of supporting the role of TGFβ in cancer and the utility of immune surveillance and antitumor immunity [4, 5]. targeting the TGFβ pathway [1]. Galunisertib (LY2157299 Systemic TGFβ ligand levels are often elevated in can- monohydrate) is an oral small molecule inhibitor (SMI) of cer patients compared to healthy individuals, and in- the TGFβ receptor I (TGFβRI) kinase that specifically creased ligand levels have been further associated with downregulates the phosphorylation of SMAD2, abrogating aggressive disease and poor prognosis [6, 7]. Elevated activation of the canonical pathway [1] (Yingling et al., TGFβ ligand levels are observed in patients whose [25]). By targeting TGFβRI, signaling via all three TGFβ tumor cells are both sensitive (i.e. receptor positive, ligands is blocked [1]. Galunisertib demonstrates the abil- TGFβ ligand dependent) or insensitive (i.e receptor ity to inhibit TGFβ-dependent tumor cell intrinsic and negative, TGFβ ligand independent) to TGFβ signaling. extrinsic functions in vitro and in vivo, and to inhibit Furthermore, aberrant TGFβ signaling has been impli- tumor-cell growth in established tumor mouse models cated in several human diseases, including malignancies (Yingling et al., [25]). Galunisertib is currently under clin- such as glioblastoma and breast cancer [8–10]. ical development in combination with checkpoint inhibi- TGFb additionally plays a non-redundant, crucial role tors (including nivolumab and durvalumab) in patients in regulating immunity. TGFβ is produced by a number with NSCLC, HCC, or pancreatic cancer (NCT02423343; of immune cells and plays an essential role in the regula- NCT02734160). tion of immune responses and immune tolerance [4, 11]. In the current study, we set out to characterize in Genetic deletion and antibody neutralization studies detail the impact of galunisertib-mediated TGFβR1 have demonstrated that TGFβ inhibition enhances T cell blockade on anti-tumor immunity. Using both in vitro [12] and NK cell differentiation and function [13], sug- and in vivo model systems, we show that galunisertib gesting that pharmacologic inhibition of TGFβ signaling enhances the development of anti-tumor T cell immun- might decrease the suppression of host immune surveil- ity through modulating both effector and regulatory T lance. Furthermore, deletion of TGFβ signaling in myeloid cell function. Using an immunogenic 4 T1-LP breast cells has been shown to enhance their anti-tumorigenic tumor model, we show that galunisertib mediates robust properties [14]. The immunological consequences of anti-tumor T cell immunity and promotes the establish- TGFβ antagonism are particularly relevant in the context ment of T cell memory and antigen spreading. Using in of anti-tumor immunotherapy, and blockade of the TGFβ vitro assays and primary human Treg cells we show that pathway has become an attractive approach to inhibit the Galunisertib treatment blocks the suppressive activity of multitude effects the TGFβ pathway has on cancer human Tregs, further highlighting its important role in Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 3 of 15 T cell immunity. The TGFβ pathway was recently de- after 5–7 days of culture. Rescue of proliferation was scribed as a potential mechanism of resistance for anti- calculated according to the formula: Percent recovery PD-1/L1 checkpoint blockade [26, 27]. To this end, we of proliferation = (%CFSE low T naive in treated Treg show that galunisertib treatment at a clinically relevant co-culture - %CFSE low T naive in untreated Treg dose enhances the anti-tumor activity of anti-PD-L1 co-culture)/(% CFSE low untreated T naive monocul- resulting in robust tumor regressions associated with en- ture stimulated with beads - %CFSE low T naive in hanced T-cell activation signatures, further supporting untreated Treg co-culture) × 100%. One-way ANOVA the clinical development of targeting TGFβRI in com- followed by Dunnett’s test was performed to assess bination with checkpoint blockade. Clinical trials evalu- statistical significance. ating galunisertib in combination with anti-PD-1 immunotherapy are currently being conducted (https:// Murine cell lines clinicaltrials.gov; NCT02734160 and NCT02423343) and CT26.WT (CT26) colon and 4 T1 and EMT6 breast thus, gives this research a highly translational impact. tumor lines, were purchased from American Type Cul- ture Collection (ATCC; Manassas, VA). MC38 colon Methods tumor cell line was purchased from the NCI tumor re- Human CD8 T cell suppression assays with TGFβ pository (Frederick, MD). The 4T1 luciferase positive CD8 T cells were purified from healthy donor blood (4T1-LP) cell line was developed at Lilly NYC from the (New York Blood Center, NY, NY) with RosetteSep 4T1 parental cell line stably transduced with firefly lucif- Human CD8 T cells enrichment kit (Stemcell erase (luciferase plasmid pLXSN-luc, G418). The EMT- Technologies) and labeled with 1 mM CFSE (Invitrogen) 6-LM2 was generated following serial passage of meta- in pre-warmed PBS+5%FCS for 10 min at 37 °C. Cells static parental EMT6 cells [13]. were then plated onto 96-well plates (5 × 10 /well) in complete RPMI media (Gibco) and stimulated with Mice −/− human T cell activation/expansion beads (Miltenyi Female Balb/c (WT and Rag )and C57BL/6mice(6to Biotech). Cells were cultured with or without TGFβ1at 8 weeks of age) were purchased from Harlan Laboratories/ 10 ng/ml. Galunisertib was added at indicated Envigo. All experimental procedures were done in concentration (0.1μMto10 μM) with DMSO as vehicle accordance with the guidelines of the NIH “Guide for Care control. Percent CD8 T cell proliferation was measured and Use of Animal” and approved protocols reviewed by by assessing CFSE dilution by FACS (BD LSRFortessa) Institutional Animal Care and Use Committee. after 5 days of culture. Recovery of T cell proliferation was calculated according to the formula: % of Max In vivo studies: Tumor challenge and treatment proliferation = % CFSE low of sample/(average of CFSE experiments low for control with no TGFβ). One-way ANOVA 4T1 and 4T1-LP tumors were generated by injection of followed by Dunnett’s test was performed to assess stat- 1×10 cells orthotopically in the mammary fat pad of istical significance. Balb/C mice. Galunisertib was dosed P.O. at 37.5 mg/kg, 75 mg/kg or 150 mg/kg twice daily (BID) for 28 days, Human Treg suppression assay with HEC (1% hydroxyethyl cellulose (HEC) in 25 mM CD4 cells purified from heathy donor blood (New York phosphate buffer, pH = 2) as control vehicle. For + 6 5 Blood Center, NYC) using the Rosetta CD4 T cell combination therapy studies, 1 × 10 CT26 or 5 × 10 enrichment kit (Stem Cell Technologies). CD25 and MC38 cells were injected subcutaneously into the flank − + CD25 T cells were then isolated using human CD25 T of Balb/c or C57BL/6 mice, respectively. Galunisertib cell microbeads (Miltenyi). Naïve CD25 T cells were was dosed at 75 mg/kg BID for 21 days and anti-PD-L1 labeled with 1 mM CFSE (Invitrogen) as described antibody (clone 178G7; Lilly NYC) or Rat IgG antibody − + above. CD25 naïve T cells and CD25 Tregs were re- was given 3 times intraperitoneal at 500μg/dose every suspended in complete RPMI media (Gibco) and plated 7 days (q7dx3). For depletion of CD8 T cells, mice onto 96-well plates at indicated ratios of Treg cells to were injected i.p. with 200 μg of CD8a antibody naïve T cells with 5 × 10 cells/well in total; except for (clone 53–6.7; eBioscience) on day 1, 2 and 3 after Tregs alone and untreated naïve T cells which were tumor challenge, followed by injection of 200 μg plated at 2.5 × 10 cells/well. Cells were then stimulated weekly throughout the experiment. For all studies, with CD3/CD28/CD2 antibody coated beads (Miltenyi) mice were randomized by body weight or tumor vol- at a bead to cell ratio of 1:1with unstimulated CD25 ume into groups of 8–15 mice prior to treatment. For naïve T cells as a control. Galunisertib (0.1μMto MOA experiments, separate subgroups of 3–5ani- 10 μM) was added with DMSO as vehicle control. mals/MOA timepoint were pre-assigned at study initi- Proliferation was measured by CFSE dilution as above ation and not included in survival evaluation. Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 4 of 15 Tumor volume was calculated using a formula: Tumor background MFI of blank well; “Adjusted Net MFI”, cal- 3 2 Volume (mm )= π/6 * Length * Width . Animals were culated: if MFI > lower limit of detection (LLOD, back- sacrificed due to progressive disease if tumor burden ground MFI + 3 standard deviations), then “Adjusted was greater than 2500 mm and if growth would surpass Net MFI” = “Net MFI”, if MFI < LLOD, then “Adjusted 2500 mm before the next scheduled measurement. For Net MFI” = LLOD – background. Adjusted Net MFI was rechallenge experiments mice with complete regressions used to calculate relative gene expression normalizing (tumor volume < 14 mm ) were rechallenged as each gene to the geometric mean of the MFI of selected indicated and followed for ~ 30 days. housekeeping genes (HKG) (adjusted net MFI/geometric Tumor volumes compared to control (%T/C) were mean HKG MFI) multiplied by a scaling factor of 100. calculated as %T/C = 100 x ΔT/ΔC, whereby ΔT= Data visualizations were done using TIBCO Spotfire® mean tumor volume of treated group, and ΔC = mean software (Spotfire, Somerville, MA). tumor volume of the control (vehicle) on indicated dayminus themean tumor volumeonthe baseline. Results Statistical analysis was performed by two-way re- Galunisertib blocks TGFβ1 mediated suppression of naïve peated measures analysis using the log transformation T cell proliferation and blocks Treg mediated suppression of tumor volume. Predefined pairwise comparisons of naïve T cells were conducted as indicated. TGFβ signaling plays an important role in suppressing an immune reaction and inducing tolerance. In particu- Isolation of tumor-infiltrating cells and lymphoid tissue lar, TGFβ signaling inhibits innate and adaptive immune cells functions and induces suppressive immune cells. To test Tumors and spleens were harvested from individual if galunisertib could rescue TGFβ suppressed immune mice at specific MOA time points after tumor cell in- cell subsets, naïve T cell suppression assays were estab- oculation. Single cell suspensions were made by hom- lished, and suppression mediated by TGFβ1orbyT ogenizing each tissue separately through 40 μm nylon regulatory cells (Tregs) was tested in in vitro culture sys- mesh strainers into complete media (RPMI+ 10% FBS). tems. For these experiments, naïve human CD8 T cells After RBC lysis (ACK lysis buffer; Gibco) when required, were stimulated with anti-CD3/anti-CD28 beads in the all samples were washed and re-suspended in FACS buf- presence or absence of TGFβ1. As shown in Fig. 1a, fer (PBS + 4% BSA) for fresh FACS analysis or snap fro- while TGFβ1 potently suppressed the proliferation of zen for gene expression analysis. CD8 T cells, addition of galunisertib resulted in a dose- dependent rescue of proliferation in the TGFβ1treated FACS analysis cultures, with enhanced proliferation observed at the Single cell suspensions prepared from mouse tumors higher doses of galunisertib. To evaluate the ability of and spleens were pre-incubated with 1 μl/ml anti-CD16/ galunisertib to modulate Treg suppressive activity, + + 32 monoclonal antibody (Fc block; Tonbo) for 30 min at CD4 CD25 Treg cells were co-cultured with naïve T cells + − 4 °C and then stained with indicated fluorochrome- (CD4 CD25 ) in the presence of anti-CD3/anti-28/anti- + + conjugated antibodies (eBioscience) and a fixable CD2 stimulation. While CD4 CD25 Tregs potently viability dye (Life Technologies). Labeled cells were suppressed naïve T cell proliferation, addition of acquired BD LSRFortessa and data processed using galunisertib fully reversed the suppression of proliferation, FlowJo software (Treestar). demonstrating a role for galunisertib in reversing Treg mediated immune suppression (Fig. 1b). Quantigene® gene expression analysis Total RNA was isolated from snap frozen tumor tissue Galunisertib monotherapy induces regression of lysed using the MagMax™ 96 Total RNA isolation kit immunogenic 4T1-LP tumors (Life Technologies) homogenized with steel beads on a To explore the impact of galunisertib monotherapy on TissueLyser (Qiagen) for 2 min at 25 Hz. Samples were preventing growth of established tumors, we utilized the processed washed, and incubated with DNase, on the poorly immunogenic murine triple negative breast MagMax™ Express 96 Processor. 500 ng of RNA was tumor model, 4T1, and a variant engineered to express incubated in duplicate with QuantiGene® magnetic cap- luciferase, (4T1-LP). For these experiments, immune ture beads, probesets, and blocking reagent (Affymetrix) competent Balb/c mice were injected orthotopically in and analyzed on the FlexMap 3D® (ThermoFisher, Wal- the mammary fat pad with 4T1-LP or 4T1 tumors. tham, MA). Level of RNA detection was determined by When tumors were well established (~300mm ,~8– mean fluorescence intensity (MFI) and converted into 11 days after implantation), animals were treated with adjusted net MFI using an in-house quality control ana- galunisertib at 75 mg/kg BID. Animals were treated for lysis script. “Net MFI”: sample was calculated as MFI – 28 days then followed for tumor growth. In the 4T1-LP Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 5 of 15 ab ns + TGF + Tregs Fig. 1 Galunisertib blocks TGFβ1 mediated suppression of naïve T cell proliferation and blocks Treg mediated suppression of naïve T cells. a Human CD8 cells purified from healthy donor whole blood where labeled with CFSE and stimulated with anti-CD3/CD28 beads in the presence of TGFβ1 and galunisertib. CD8 T cell proliferation was measured by assessing CFSE dilation by flow cytometry after 5 days of culture. Percent recovery of CD8 T cell proliferation at different concentrations of galunisertib (0-10 μM) as indicated and representative histograms are shown. + + DMSO was used as vehicle control. Data shown are combined data of 4 healthy donors. b Human CD4 CD25 cells purified from healthy donor + − whole blood where labeled with CFSE and stimulated with anti-CD3/CD28 beads in the presence of galunisertib. Autologous CD4 CD25 Treg + − were added at a 1:1 ratio of Treg cells to naïve T cells. Naïve CD4 CD25 T cell proliferation was measured by assessing CFSE dilation by flow + − cytometry after 5–7 days of culture. Percent recovery of CD4 CD25 T cell proliferation at different concentrations of galunisertib (0-10 μM) as indicated and representative histograms are shown. Data shown are combined data of 4 healthy donors; representative of 3 independent experiments. One-way ANOVA with Dunnett’s test was used to compare the galunisertib treatments to the DMSO treatment. ****: p ≤ 0.0001; ***: p ≤ 0.001; **: p ≤ 0.01; *: p ≤ 0.05; ns: p ≥ 0.05 model, the majority of mice (10/12) responded to galuni- eventually progressed without treatment. The spontan- sertib therapy, including 4/12 complete responses (Fig. eous activity we observe in a few of the 4T1-LP control 2a); in contrast, none of the poorly-immunogenic 4T1 mice (Fig. 2a) is likely reflective of an immune response bearing mice responded to galunisertib therapy (Fig. 2b), to the implanted tumors, and this immune response suggesting that the presence of a foreign antigen (i.e. LP) may in fact be the mechanism by which galunisertib is , potentially enhanced the ability of galunisertib to in- so much more active as a monotherapy in 4T1-LP com- duce the rejection of the 4T1-LP derivative. In a previ- pared to 4T1 parental. We speculate that the immuno- ous study, a survival benefit advantage with galunisertib genic nature of 4T1-LP is likely what impacts was observed in the poorly-immunogenic 4T1 tumor galunisertib monotherapy activity in this model, while in model (Yingling et al., [25]). This may reflect the early less immunogenic tumor models, combination with start of treatment in that study (day 4 after tumor im- anti-PD-L1 is needed (described below). plantation compared to 8–11 days in the study pre- To further evaluate and interrogate the impact of sented here) or it may be a result of an anti-metastatic galunisertib on anti-tumor activity, mice bearing 4T1- activity rather than an effect only on primary tumor LP tumors in the mammary fat pad were treated for growth. 28 days (starting at day 8 post tumor challenge) with A few mice in the vehicle control group in the 4T1-LP three different doses of galunisertib (37.5, 75 and model, but not in the parental 4T1 model, showed an 150 mg/kg BID). Anti-tumor activity was observed at initial tumor response before eventually progressing (Fig. all 3 doses of galunisertib, with a dose dependent in- 2a), suggesting that spontaneous responses to immuno- crease in activity as assessed by both mean tumor genic tumor cell lines can occur in some mice. This may volume and CR (Fig. 3a-d). Anti-tumor activity was reflect the different T cell repertoire between individual observed following an initial growth pattern similar to mice as we did not use TCR transgenic mice, or suggest- controls, indicating a delayed response to treatment ing that these mice developed an immune response to a and possible immune mediated mechanism. Following dominant CTL epitope of LP, which may lead to reduced cessation of therapy on day 36 (28 days of treatment), tumor invasiveness and spontaneous regression. responding mice progressed to complete responders [28]. Although, a few mice of the control group in a dose-dependent manner, with 1/10 CR in mice showed initial spontaneous activity, all untreated tumors treated with 37.5 mg/kg BID, 3/10 CRs in mice % of Max Proliferion Compared Control (+/- SEM) Control DMSO Gal 0.1µM Gal 0.3µM Gal 1µM Gal 3µM Gal 10µM % Rescue Naive T cell Proliferation Naive T cells DMSO Gal 1.1µM Gal 3.3µM Gal 10µM Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 6 of 15 Importantly, PK/PD profiling studies of galunisertib 4T1-LP Model suggest that administration of 75 mg/kg BID in preclin- Individual Tumor Growth Curves ical models or 150 mg/kg BID in patients can achieve significant target modulation in vivo over a 24-h period [1, 29]. Thus, we show anti-tumor activity with galuni- sertib at clinically relevant doses. To begin to understand how galunisertib treatment modulated immune cells within the tumor, tumors from mice galunisertib treated or vehicle control mice were harvested 8 days after therapy initiation and the changes in T cell infiltration were analyzed by flow cytometry. Control Relative to control animals, a modest increase in both Galunisertib CD3 T cells, mainly CD8 T cells, and NK cells was observed in tumors of mice treated with the clinically 020 40 60 80 relevant dose of 75 mg/kg galunisertib (Fig. 3c), indicat- Gal (BID): ing a role of galunisertib on T-cell expansion or T-cell Day Post Tumor Challenge trafficking to the tumor site. These differences did not reach statistical significance. No significant changes were 4T1 Parental Model observed in the myeloid compartment in tumors of galu- Individual Tumor Growth Curves nisertib treated mice compared to control treated mice in this model. However, only the number of myeloid cells was analyzed and not the function; thus, whether galunisertib induces reprogramming toward an antitu- mor phenotype was not explored. This may also reflect the time point of tumor collection. To this end, a prior study with anti-mouse TGFβRII showed modulation of 500 MDSCs by blocking the TGFβ signaling pathway [13]. Control Galunisertib Galunisertib monotherapy induces immunologic memory 100 and demonstrates antigen spreading 4T1-LP tumor bearing mice that completely responded 020 40 60 80 to galunisertib therapy remained tumor free for up to Gal (BID): 85 days (49 days after treatment completed) (Fig. 3a), Day Post Tumor Challenge indicating a durable response. To test the ability of galu- Fig. 2 Galunisertib monotherapy induces regression of nisertib to induce immunologic memory to 4T1-LP tu- immunogenic 4T1-LP variant breast tumors. Mean and individual mors, mice which had completely regressed 4T1-LP tumor growth curves for Balb/c mice injected orthotopically in the mammary fat pad with 4T1-LP (a) or parental 4T1 (b) tumor cells were re-challenged orthotopically with 4T1-LP on the and treated with galunisertib (75 mg/kg BID) when tumors reached opposite flank of the original tumor injection site and ~300mm (6–8 days after implantation). The number of mice/group additionally received a primary challenge of a different rejecting tumors (complete responders, CRs) was: control (0/10 mice) triple negative breast cancer tumor, EMT6-LM2, on the and galunisertib (4/12) for 4T1-LP, and control (0/10 mice) and flank of the original tumor injection site. In all mice galunisertib (0/12) for 4T1 parental as indicated. Data shown are representative of two independent experiments with tested, complete responders rejected the re-challenge 10–12 mice/group with the 4T1-LP tumors (Fig. 3d, left panel), but did not reject EMT6-LM2 tumors (Fig. 3d, middle panel), dem- treated with 75 mg/kg BID, and 5/10 CRs observed in onstrating immunologic memory to the 4T1-LP tumor mice treated with 150 mg/kg BID (Fig. 3a). CR mice cells, but not the heterologous tumor. To evaluate the remained tumor free for an additional 49 days in the potential for epitope spreading as a result of galunisertib absence of further treatment. These data indicate that anti-tumor activity, mice which had completely galunisertib induces a potent, dose-dependent durable regressed 4T1-LP after being treated with 75 mg/kg of anti-tumor response. Metastases to lungs were not galunisertib were re-challenged in a separate experiment observed in this tumor model. with the poorly immunogenic parental 4T1 tumors, Treatments with galunisertib were well-tolerated which lack the immunogenic LP transgene and is not re- with no body weight loss observed with any of the sponsive to de-novo galunisertib monotherapy (Fig. 2b); doses tested (Fig. 3b). in all mice tested, 4T1-LP complete repressors also Tumor Volume mm Tumor Volume mm Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 7 of 15 Mean 4T1-LP Tumor Volume Galunisertib Treatment Control 37.5 mg/kg 75 mg/kg 150 mg/kg 0 1020 304050 60 Gal (BID): Days Post Tumor Challenge Galunisertib 37.5mg/kg Galunisertib 75mg/kg Galunisertib 150mg/kg 2500 2500 2500 Control Control Control 37.5mg/kg 75mg/kg 150mg/kg 2000 2000 2000 1500 1500 1500 1000 1000 1000 500 500 500 0 0 0 0 10203040 50607080 0 10203040 50607080 0 10203040 50607080 Gal (BID): Gal (BID): Gal (BID): Days Post Tumor Challenge Days Post Tumor Challenge Days Post Tumor Challenge b Mean Body Weight d Galunisertib Treatment 4T1-LP Rechallenge EMT6-LM2 Challenge 30 5000 5000 Control Galunisertib 37.5 mg/kg Galunisertib 37.5 mg/kg 37.5 mg/kg Galunisertib 75 mg/kg Galunisertib 75 mg/kg 75 mg/kg Galunisertib 150 mg/kg Galunisertib 150 mg/kg 150 mg/kg 3000 3000 10 0 0 10 203040 5060 7080 82 85 88 91 94 97 100 103 106 109 82 85 88 91 94 97 100 103 106 109 Gal (BID): Days Post Primary Tumor Challenge Days Post Primary Tumor Challenge Days Post Tumor Challenge Galunisertib (mg/kg): CD45 intra-tumor c e Parental 4T1 Rechallenge Parental 4T1 0 37.5 75 150 Individual Tumor Growth Naive Mice 2000 2000 0.88% 3.37% 1.27% 3.14% 0.81% 3.05% 0.51% 1.69% 400 400 NK Cells (DX5) 0 0 85 90 95 100 105 110 85 90 95 100 105 110 Day Post Tumor Challenge Day Post Tumor Challenge Fig. 3 (See legend on next page.) T cells (CD3) Mean Tumor Volume mm (+/-SEM) Tumor Volume mm Mean body weight (g) (+/- SEM) Tumor Volume mm 3 3 Tumor Volume mm Mean Tumor Volume mm (+/-SEM) Rechallenge Tumor Volume mm 3 3 Tumor Volume mm Mean Tumor Volume mm (+/-SEM) Rechallenge Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 8 of 15 (See figure on previous page.) Fig. 3 Galunisertib monotherapy displays dose dependent activity against established orthotopic 4T1-LP tumors with immunologic memory and antigen spreading. a Mean and individual tumor growth curves for Balb/c mice injected orthotopically in the mammary fat pad with 4T1-LP tumor cells and treated with galunisertib (37.5, 75 or 150 mg/kg BID) when tumors reached ~ 300 mm3 (8 days after implantation). The number of CRs were: 1/10, 3/10 and 5/10 for mice treated with Galunisertib at 37.5 mg/kg, 75 mg/kg and 150 mg/kg, respectively. b Percent body weight change on average of mice treated with galunisertib at 37.5 mg/kg, 75 mg/kg and 150 mg/kg. c Frequencies and representative plots of CD3 and NK T cells of total live CD45 cells in in single cell suspensions prepared from tumors harvested after 8 days of galunisertib treatment. Tumors from 5 mice/group treated with 75 mg/kg galunisertib or vehicle control were analyzed. Student’s t-tests were used to evaluate statistical significance (ns: p ≥ 0.05). d Mice which had regressed 4T1-LP tumors after treatment with galunisertib (37.5, 75 or 150 mg/kg BID for 28 days; as shown in a) were rechallenged orthotopically with 4T1-LP in one mammary fat pad and received a primary challenge of a different triple negative breast cancer tumor, EMT6-LM2, in an alternate fat pad, at day 85 post the original 4T1-LP tumor challenge. Average tumor growth curves of secondary 4T1-LP and EMT-6-LM2 challenge are shown for each group. e Mice which had regressed 4T1-LP tumors after treatment with galunisertib (75 mg/kg BID for 28 days) were rechallenged orthotopically with the poorly immunogenic 4T1 parental tumor cells at day 85 post the original 4T1-LP tumor challenge. Average tumor growth curves of secondary 4T1-LP challenge are shown. Individual tumor growth curves of naïve mice injected orthotopically with 4T1 parental tumors are shown as a control. Data shown are representative of two independent experiments with 10–12 mice/group rejected the parental 4T1 challenge (Fig. 3e, right panel), tumors indicating a requirement for an adaptive immune demonstrating the potential for galunisertib anti-tumor system, and in particular CD8 T cells, in this model activity to mediate antigen spreading. (Fig. 4). Galunisertib anti-tumor activity in the 4T1-LP model is Combined blockade of TGFβR1 and PD-L1 enhnaces CD8 T cell dependent regression of tumors The delayed response to galunisertib and the modest in- Because the in vitro and in vivo galunisertib monother- crease in tumor-infiltrating lymphocytes (TILs) in apy data strongly suggested that galunisertib was able to treated mice suggested that the adaptive immune re- modulate anti-tumor T cell immunity, we investigated sponse may be involved in the mechanism of tumor re- whether galunisertib could synergize with PD-L1 check- jection following galunisertib therapy. To evaluate the point blockade and result in improved tumor regres- role of the adaptive immune response in galunisertib sions. Anaphylactic reactions have been reported with anti-tumor activity, studies using the orthotopic 4T1-LP PD-L1 and PD1 monoclonal antibodies in the ortho- −/− model were carried out in the RAG mice or in Balb/c tropic 4T1 tumor model [30] (Mall et al., [31]), thus, for mice depleted of CD8 T cells by treatment with an these studies we utilized the CT26 mouse model well −/− immune depleting anti-CD8α antibody. In both RAG known to be responsive to various degrees to PD-1 axis and CD8 T cell depleted mice bearing 4T1-LP tumors, immunotherapy [32]. Balb/c mice were injected with galunisertib therapy was unable to induce regression of CT26 tumor cells and treated with galunisertib, anti-PD- ab RAG-/- Mice CD8α Depleted Mice Control Control Galunisertib 75mg/kg Control CD8α depleted 1500 Galunisertib 75mg/kg Galunisertib CD8α depleted 0 5 10 15 20 0 5 10 15 20 Gal (BID): Gal (BID): Days Post Tumor Challenge Days Post Tumor Challenge −/− Fig. 4 Galunisertib anti-tumor activity in the 4T1-LP model is CD8 T cell dependent. a Mean tumor growth curves for Rag mice injected orthotopically in the mammary fat pad with 4T1-LP tumor cells and treated with galunisertib (75 mg/kg BID × 28 days) when tumors reached ~ 300 mm3 (8 days after implantation). b Mean tumor growth curves for Balb/c mice injected orthotopically in the mammary fat pad with 4 T1-LP tumor cells and treated with galunisertib (75 mg/kg BID × 28 days) plus depleting antibody for CD8α (200 μg/mouse). Treatment was initiated when tumors reached ~ 300 mm3 (8 days after implantation). CD8α monoclonal antibody was injected on day 1, 2 and 3 after tumor challenge, followed by injection weekly throughout the experiment. Data represent two independent experiments with 8 mice/group Mean Tumor Volume mm (+/-SEM) Mean Tumor Volume mm (+/-SEM) Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 9 of 15 L1 or a combination of both for 21 days. Treatment was active in this setting with 5/15 and 3/14 CR respectively initiated on Day 6 when tumors were ~100mm and while the combination of anti-PD-L1 and galunisertib continued for 28 days. The mean tumor growth under therapy resulted in marked enhancement of response (9/ each condition is presented in Fig. 5 (left panel), and 14 CRs) and all animals responding to treatment (Fig. 5). shows that while both galunsertib and anti-PD-L1 ther- There was a significant antitumor benefit with the com- apy were active in this model, the combination of galuni- bination group versus each monotherapy (p < .001). To sertib and anti-PD-L1 resulted in enhanced anti-tumor test if each treatment could result in immunologic mem- activity. As shown in the individual animal plots, anti- ory, mice with CRs were re-challenged with CT26 PD-L1 or galunisertib monotherapy were modestly tumors 85 days after primary tumor challenge (51 days Mean CT26 Tumor Volume Galunisertib and Anti-PD-L1 Therapy Rat IgG Anti-PD-L1 Galunisertib Combo 0 5 10 15 20 25 Gal (BID): Anti-PD-L1: Days Post Tumor Challenge Galunisertib Monotherapy Galunisertib and Anti-PD-L1 Combination Therapy Anti-PD-L1 Monotherapy 3000 3000 Rat IgG Rat IgG Rat IgG Galunisertib Combo PD-L1 2500 2500 2500 2000 2000 1500 1500 1000 1000 500 500 0 0 0 102030405060 0 102030405060 0 1020 3040 506070 8090 100 Gal (BID): Anti-PD-L1: Gal (BID): Anti-PD-L1: Days Post Tumor Challenge Days Post Tumor Challenge Days Post Tumor Challenge b c Mean MC38 Tumor Volume Secondary CT26 Rechallenge Galunisertib and Anti-PD-L1 Therapy Rat IgG Galunisertib * Anti-PD-L1 Anti-PD-L1 Galunisertib Combo Combo 80 85 90 95 100 105 0 5 10 15 20 Days Post Primary Tumor Challenge Gal (BID): Anti-PD-L1: Day Post Tumor Challenge Fig. 5 Combined blockade of TGFβR1 with Galunisertib and PD-L1 induce robust regression of murine colon tumors. Mean and individual tumor growth curves for Balb/c mice injected with CT26 tumor cells and treated with galunisertib (75 mg/kg BID for 28 days) and/or anti-PD-L1 antibody (500 μg/dose, q7dx3) when tumors reached ~100mm (6 days after implantation). The percentages of CRs were: control (0/15 mice), galunisertib monotherapy (3/14), anti-PD-L1 monotherapy (5/15) and combination therapy (9/14). b Mice which had regressed CT26 tumors after treatment with galunisertib and/or anti-PD-L1 were rechallenged with CT26 tumor cells on the contralateral flank at day 85 post the original tumor challenge. Individual tumor growth curves of secondary CT26 challenge are shown for each treatment group. c Mean and individual tumor growth curves for C57BL/6 mice injected with MC38 tumor cells and treated with galunisertib (75 mg/kg BID for 21 days) and/or anti-PD-L1 antibody (500 μg/dose, q7dx3). Treatment was initiated 3 days after tumor cell implantation. Data shown are representative of two independent experiments with 10–12 mice/group 3 3 Tumor Volume mm Mean Tumor Volume mm (+/-SEM) Tumor Volume mm Rechallenge Tumor Volume mm Mean Tumor Volume mm (+/-SEM) Tumor Volume mm Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 10 of 15 after initial treatment cessation). All complete re- mice were collected day 10, 16, and 22 after tumor chal- sponders in monotherapy and combination treatment lenge (i.e. 4, 10, and 16 days after initiation of therapy) groups rejected the re-challenge with the CT26 tumors and subjected to high-content molecular profiling using TM (Fig. 5b, left panel); however, one animal from the com- a custom designed Quantigene gene panel to detect T bination cohort, defined as a long-term partial responder cell activation and intra-tumoral inflammation (Table 1). (indicated by * in Fig. 5a right panel) was unable to reject Galunisertib monotherapy (75 mg/kg) did not appre- secondary tumor challenge. The ability of galunisertib to ciably alter the set of immune genes analyzed relative to enhance the activity of anti-PD-L1 immunotherapy was control tumors at any time point evaluated (Fig. 6, top confirmed in the MC38 tumor model, which is historically panel). Anti-PD-L1 monotherapy resulted in an en- less responsive to checkpoint immunotherapy and consid- hanced T cell infiltration and activation profile, exempli- ered to be more myeloid biology driven [32]. In this fied by the increase in multiple immune activation and model, where treatment began on day 3 after tumor chal- inflammation transcripts such as Ccl5, Itgax, Icam1, lenge, similar monotherapy and combination therapy ac- Foxp3, Lag3 by day 22 (Fig. 6a). On the other hand, the tivity was observed albeit with more moderate activity combination treatment demonstrated an early signature overall (Fig. 5c). of enhanced T cell activation and inflammation exempli- fied by the upregulation of transcripts for Ifnγ, Lag3, Combination of galunisertib and anti-PD-L1 checkpoint Ccl3, Ccl4, Ccl5, and Tnfrsf18) beginning on Day 16 blockade induces an intra-tumor immune related gene (after only 10 days of therapy, where only a minor expression profile that is accelerated and enhanced change in activation was detected with anti-PD-L1 compared to anti-PD-L1 monotherapy monotherapy), and continuing at day 22 with an en- To further elucidate the mechanism of action of the hanced gene expression related to T cell infiltration combination activity of galunisertib and anti-PD-L1, (Ptprc, Cd8b1, Cd3e, and Cd4) and T cell activation and gene expression studies were carried out on tumors inflammation (Il2, Il4, Il17a, Lag3, Ifnγ, Ifnα, Ifnβ1, from CT26 tumor bearing mice treated with control, Foxp3, Cd274, and Pdcdlg2) (Fig. 6a). The gene profile anti-PD-L1, galunisertib or a combination of anti-PD-L1 for the combination cohort was similar but more robust plus galunisertib. For these studies, tumors from treated compared to PD-L1 monotherapy detected at the later Table 1 Custom designed Quantigene™ gene panel to detect T cell activation and intra-tumoral inflammation Cell type-specific Coinhibitory & Cytokines & Immunosuppressive Markers of Angiogenesis; EMT TGFb HKGs markers Costimulatory Chemokines Enzymes T cell activation Endothelial activation markers pathway Cd14 4632428N05Rik (Vista) Ccl2 (MCP-1) Arg1 Cd69 Cdh5 Cdh1 Smad4 Gus Cd3e Cd200r1 Ccl3 (MIP-1a) Ido1 Gzmb Hif1a Epcam Tgfb1 Hprt Cd4 Cd274 (PD-L1) Ccl4 (MIP-1b) Mpo Ifng Vcam1 Fap Tgfb2 Ppib CD68 Cd40lg Ccl5 (RANTES) Nos2 Vegfa Snai1 Tgfb3 Rps18 Cd8b1 Cd86 Csf2 (GM-CSF) Tdo2 Vegfc Twist1 Tgfbr1 Foxp3 Havcr2 (TIM3) Cxcl1 Icam1 (CD54) Vim Tgfbr2 Itgam (CD11B) Icos Ifna2 Sele Cspg4 Itgax (CD11C) Lag3 Ifnb1 Klrk1 Pdcd1 (PD-1) Il10 (CSIF) Ms4a1 Pdcd1lg2 (PD-L2) Il13 Ptprc (CD45) Pvr Il17a Pvrl2 Il2 Tigit Il4 Timd4 (TIM4) Il5 Tnfrsf18 (GITR) Il6 Tnfrsf4 (OX40) Il9 Tnfrsf9 (4-1BB) Tnf Tnfsf18 (GITRL) Tnfsf4 Tnfsf9 (CD137L, 4-1BBL) Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 11 of 15 Fig. 6 Combination of galunisertib and anti-PD-L1 checkpoint blockade induces an intra-tumor immune related gene expression profile that is accelerated and enhanced compared to anti-PD-L1 monotherapy. Log2 Fold Change in gene expression of galunisertib (75 mg/kg BID for 28 days) and/or anti-PD-L1 (500 μg/dose, q7dx3) treated CT26 tumors against vehicle control. Differential expression and p-value was evaluated by two-way ANOVA testing. Labelled and highlighted genes are log2FC > 1, p-value< 0.05, are shown in Volcano Plots (a) and Venn Diagrams (b). Tumors were harvested and analyzed at different time points after tumor challenge: day 10, 16 and 22 (treatments were initiated at 6 days after implantation) time point (day 22) (Fig. 6a and b). Interestingly, the treat- during therapy, and T cell frequencies were similar for ments also resulted in increased expression of some im- monotherapy PD-L1 and combination therapy (data not munosuppressive genes, such as Ido1, Mpo, Nos2 and shown), suggesting that the effects of combination were Tdo2, which may reflect a counter-regulatory mechanism modulated at the effector function level. Finally, we ob- induced by the tumor and/or myeloid cells in response to served that some genes such as FAP were upregulated at enhanced IFNγ production by anti-PD-L1 or combination day 22 upon anti-PD-L1 treatment but not with combin- treated tumor-infiltrating T cells. In support of this, pre- ation therapy, suggesting that galunisertib may be acting clinical work using murine tumor models have shown that by remodeling tumor stroma (Fig. 6a), as previously de- dual targeting of IDO and checkpoints results in enhanced scribed in the literature with other inhibitors of the TGFβ anti-tumor immunity [33–35]. Overall, combination ther- pathway [13, 36]. apy resulted in an accelerated and more robust increase in genes indicative of T cell activation compared to either Discussion monotherapy suggesting that inhibiting immune suppres- TGFβ plays diverse and important roles in tumorigen- sion with galunisertib may accelerate the biological activity esis, including tumor proliferation, invasion and metas- of anti-PD-L1. Examination of immune cell subset fre- tasis, inflammation, angiogenesis, and escape of immune quencies in tumors by flow did not detect major changes surveillance. Therefore, targeting the TGFβ signaling Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 12 of 15 pathway has been an attractive objective for cancer ther- tumor activity in immune-competent murine tumor apy, and several drugs have been identified and are models. We show that galunisertib monotherapy induces under clinical development [37–39]. Galunisertib is a dose-dependent regression of well-established immuno- small molecule inhibitor of the kinase domain of genic 4T1-LP breast tumors. The responses were dur- TGFβRI. Binding of TGFβ ligands to TGFβRII is the first able with immunological memory as demonstrated by step to initiate activation of the TGFβ signaling pathway. rechallenge experiments with 4T1-LP tumors as well as Once bound to TGFβRII, this ligand/receptor complex a second triple negative breast tumor cell line. Of note, binds to TGFβRI to form a heterotrimeric complex. For- mice that rejected the immunogenic 4T1-LP tumors mation of this complex results in phosphorylation of the were also able to reject 4T1 parental cells upon rechal- serine/threonine kinase domains of the receptors, lenge, suggesting the development of a secondary followed by activation of the canonical SMAD2/3 signal- immune response via antigen spreading as a conse- ing pathways as well as non-canonical (i.e. MAPK) path- quence of effective tumor targeting. The anti-tumor ways [39]. These pathways modulate transcription of activity of galunisertib in the 4T1-LP tumor model was numerous target genes, resulting in a variety of effects. CD8 T cell dependent and associated with a modest By blocking the kinase domain of TGFβRI, galunisertib increase in T-cell infiltration in tumors. The increase may effectively inhibit signaling via the TGFβ pathway. was modest and did not reach statistical significance Preclinical and clinical research on galunisertib, though, which might reflect the time point chosen for including the treatment of over 800 patients, has dem- tumor collection. It is well established in the immuno- onstrated that SMIs of TGFβ can safely be developed for oncology field that the spatial distribution and location of clinical testing, provided there is an adequate under- immune cells is highly important. In fact, the recent publi- standing of the pharmacokinetic/pharmacodynamics cations combining TGFβ inhibition and PD-L1 blockade (PK/PD) relationship, as most of the toxicities in animal show that the main mechanism of action of TGFβ inhib- models that were of concern prior to the start of clinical ition is to increase T-cell infiltration into tumor [26, 27]. development of galunisertib have not been observed in In addition, using an anti-TGFβRII blocking antibody we humans [1]. Furthermore, preclinical and clinical efforts have previously shown that blocking TGFβ signaling in suggest that the biology of the TGFβ inhibition is largely the EMT6 tumor model induces immune infiltration [13]. dependent on the microenvironment, perhaps more than We did not investigate metastasis to lungs in either originally anticipated. For example, TGFβ1 is a potent the 4T1 or 4T1-LP tumor models used here. However, inducer of angiogenesis [40], by directly inducing VEGF we have previously shown that the anti-mouse TGFβRII expression [41], or recruiting other cells, such as mono- antibody significantly inhibits the growth of established cytes, which in turn secrete proangiogenic molecules 4T1-parental primary tumors and diminishes the spon- [42]. TGFβ can also manipulate the tumor microenvir- taneous pulmonary metastasis [13]. In addition, it was onment to favor the evasion of cancer cells from im- shown that galunisertib in combination with anti- mune surveillance via tampering with the antitumor CTLA4 therapy suppresses both primary melanoma functions of T cells, NK cells, B cells, and other cells tumor growth as well as metastases in a physiological [43–45]. This activity of TGFβ may be mediated through relevant trangenic melanoma model (Hanks et al. [50]). its direct effect on these cells, as well as via its ability to Furthermore, we described that galunisertib inhibits induce Foxp3 Tregs [46]. Both cancer-intrinsic and TGFβ mediated migration of U87MG glioblastoma cells immune-mediated effect of TGFβ in breast cancers have in vitro in a dose-dependent manner [51]. Notably, in been described [47–49]. Thus, a focus on direct tumor this model system, galunisertib reduced baseline migra- cell cytotoxicity may be misleading and provide incon- tion of U87MG cells in the absence of exogenous clusive observations that will not be helpful to advance TGFβ1, presumably by inhibiting autocrine signaling clinical development of future TGFβ inhibitors. Early through TGFβRI. Together this suggests that galuniser- studies using immune-compromised animals may there- tib has the capacity to suppress the development of me- fore also have limited the screening for TGFβ inhibi- tastasis and that TGFβ pathway blockade of the parental tors. It now appears that an active immune response 4T1 model is sufficient to inhibit metastasis to lung. is essential to assess the effect of TGFβ signaling in- Importantly, we have previously shown that the TGFβ hibition in animal models; thus, immune-competent pathway is abrogated upon treatment with galunisertib animal models maybemorepredictivetoevaluate both in vitro and in vivo [51]. We demonstrated that TGFβ inhibitors. Consequently, more novel preclinical galunisertib inhibited TGFβ-induced pSMAD in various testing assays are required than those traditionally tumor cell lines, including 4 T1-LP, in vitro in a dose- used in oncology research. dependent manner [51]. Furthermore, we reported a Here we describe the impact of galunisertib to modu- galunisertib time and dose-dependent inhibition of late the immune system and its ability to enhance anti- endogenous TGFβ-dependent signal transduction in vivo Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 13 of 15 in EMT6-LM2 murine syngenic tumor models [51]. gene panel tested, the dose chosen or the day of collection. These data suggest that the effects of galunisertib are Similar combination therapy activity was observed in on-target. Potential off-target effects of galunisertib are the PD-L1 insensitive tumor model, MC38, albeit with further diminished, as treatment with an anti-mouse more moderate activity overall, suggesting at least TGFβRII antibody similarly inhibits the growth of estab- additive activity with potential for synergy when tar- lished mouse 4T1 and EMT6 primary [13]. geting the TGFβ and PD-1 pathways. The anti-tumor Immunotherapeutic strategies such as immune check- activity of galunisertib was tested in a broad range of point blockade have shown significant promise for treat- murine tumor models with similar results, further ment of cancers resistant to conventional modalities, suggesting that TGFβ inhibition is immune mediated leading to Food and Drug Administration (FDA) and thus not restricted to specific tumor indications. approval in advanced melanoma, renal cell carcinoma Finally, we show that galunisertib reverses both TGFβ and non-small cell lung cancer (NSCLC) [52]. Despite and Treg mediated suppression of T cell proliferation in clinical results, even with combined checkpoint blockade human cell cultures in vitro, which further highlight the [53], therapeutic success has so far been limited to a important role of galunisertib to overcome immune sup- subset of patients, calling for identification of markers pression and promote anti-tumor immunity. predicting response, identification of resistance mecha- Taken together, the results presented here demonstrate nisms and development of combinatorial therapeutic the impact of blocking TGFβ signaling and provide a approaches. To this end, TGFβ pathway inhibition repre- strong incentive to clinically explore the potential of sents an attractive strategy with its multitude of effects galunisertib treatment to enhance the development of on cancer progression and on the immune system to en- anti-tumor T cell immunity, which may be enhanced by hance the development of anti-tumor T-cell immunity. combinations with immune checkpoint inhibitors. Our Indeed, a recent study by Powles et al., reports that lack results expand on other reports demonstrating that sys- of response to atzeolizumab (anti-PDL1) in bladder can- temic treatment with monoclonal antibodies targeting cer patients was associated with an immune-excluded the TGFβ ligands or the TGFβRII inhibit metastatic phenotype that corresponded with active TGFβ in peritu- invasion of breast cancer cells in murine tumor models moral stroma and a signature of TGFβ signaling [26]. Using [2, 13], and previous work reporting that blocking TGFβ mouse models that recapitulate the immune-excluded signaling with SMIs suppresses metastasis in murine phenotype they further show that co-administration of pancreatic cancer models [54], and enhances radiation blocking antibodies to TGFβ and PDL1 reduced TGFβ sig- response and prolongs survival in xenograft models of naling, facilitated T-cell penetration of tumors, and pro- glioblastoma [55]. voked vigorous anti-tumor immunity leading to tumor Galunisertib continues to advance in clinical trials hav- regressions. In a second recent study published by Batlle ing completed Phase I [56] and is currently under inves- and colleagues, combinatorial activity of galunisertib with tigation in several Phase I and Phase II trials. Thus far, anti-PDL1 in murine colon cancer models was recently de- galunisertib has been very well-tolerated as a first-in- scribed [27]. Combination therapy induced pronounced im- class, oral cancer therapy, and remains a promising com- mune responses which eradicated most metastases, pound in clinical development (http://clinicaltrials.gov/ prolonged recurrence-free survival, and was associated with ct2/results?term=LY2157299). Our data presented here disruption of a T-cell exclusion phenotype. These results support continued clinical development of galunisertib suggest that clinical co-administration of TGFβ and PDL1 to target tumors dependent on TGFβ-driven biology for blocking agents may provide a subset of patients more growth, metastasis, and immune evasion. Whether TGFβ favorable outcomes; however, preclinical validation was per- inhibition applies to all tumors is not clear at this time. formed with either a research-grade reagent [26]or a sig- For clinical development, patient selection tools, defining nificantly excessive amount of galunisertib (800 mg/kg BID who will most likely benefit from TGFβ inhibition, remain compared to the clinically relevant dose of 75 mg/kg a challenging question. Among others, the activity of described in [1, 29]. In agreement, we demonstrate that TGFβ inhibition appears to be dependent on immune combination of galunisertib with PD-L1 checkpoint block- function; thus, it will be important to investigate new bio- ade results in a robust regression of CT26 tumors when markers that are related to immune responses which may compared to single agents. The observed antitumor benefit help with patient selection in future studies. was associated with enhanced expression of genes indica- tive of immune activation and this gene expression profile Conclusion was accelerated compared to anti-PD-L1 monotherapy. In many advanced cancers, TGFβ ligands are overex- Galunisertib alone resulted in no alteration of any of the pressed and the outcome of signaling is diverted toward tested genes. Considering the critical role of TGFβ in can- disease progression. A concerted effort has therefore cer immunity, we speculate that this may be a result of the been to develop drugs that block TGFβ signaling for Holmgaard et al. Journal for ImmunoTherapy of Cancer (2018) 6:47 Page 14 of 15 therapeutic benefit. Galunisertib is a pharmacological 8. Ganapathy V, et al. Targeting the transforming growth factor-beta pathway inhibits human basal-like breast cancer metastasis. Mol Cancer. 2010;9:122. small molecule inhibitor of the TGFβ pathway that acts 9. 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Journal for ImmunoTherapy of CancerSpringer Journals

Published: Jun 4, 2018

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