Investigational New Drugs (2020) 38:1570–1579 https://doi.org/10.1007/s10637-020-00910-9 PHASE II STUDIES Phase 1b/2a study of galunisertib, a small molecule inhibitor of transforming growth factor-beta receptor I, in combination with standard temozolomide-based radiochemotherapy in patients with newly diagnosed malignant glioma Antje Wick 1 & Annick Desjardins 2 & Cristina Suarez 3 & Peter Forsyth 4 & Ivelina Gueorguieva 5 & Tiana Burkholder 6 & Ann Louise Cleverly 5 & Shawn T. Estrem 6 & Shuaicheng Wang 7 & Michael M. Lahn 6 & Susan C. Guba 6 & David Capper 8 & Jordi Rodon 3,9 Received: 4 December 2019 / Accepted: 7 February 2020 / Published online: 5 March 2020 # The Author(s) 2020 Summary Purpose Galunisertib, a TGF-β inhibitor, has demonstrated antitumor effects in preclinical and radiographic responses in some patients with malignant glioma. This Phase 1b/2a trial investigated the clinical benefit of combining galunisertib with temozolomide-based radiochemotherapy (TMZ/RTX) in patients with newly diagnosed malignant glioma (NCT01220271). Methods This is an open-label, 2-arm Phase 1b/2a study (N = 56) of galunisertib (intermittent dosing: 14 days on/14 days off per cycle of 28 days) in combination with TMZ/RTX (n = 40), versus a control arm (TMZ/RTX, n = 16). The primary objective of Phase 1b was to determine the safe and tolerable Phase 2 dose of galunisertib. The primary objective of Phase 2a was to confirm the tolerability and pharmacodynamic profile of galunisertib with TMZ/RTX, and the secondary objectives included determining the efficacy and pharmacokinetic (PK) profile of galunisertib with TMZ/RTX in patients with glioblastoma. This study also characterized the changes in the major T-cell subsets during TMZ/RTX plus galunisertib treatment. Results In the Phase 2a study, efficacy results for patients treated with galunisertib plus TMZ/RTX or TMZ/RTX were: median overall survival (18.2 vs 17.9 months), median progression-free survival (7.6 vs 11.5 months), and disease control rate (80% [32/40] vs 56% [9/16] patients) respectively. PK profile of galunisertib plus TMZ/RTX regimen was consistent with previously published PK data of galunisertib. The overall safety profile across treatment arms was comparable. Conclusion No differences in efficacy, safety or pharmacokinetic variables were observed between the two treatment arms. Keywords Glioblastoma . Galunisertib . Radiochemotherapy . T cells . Biomarkers Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10637-020-00910-9) contains supplementary material, which is available to authorized users. * Antje Wick 5 Eli Lilly and Company, Erl Wood, UK antje.wick@med.uni-heidelberg.de 6 Eli Lilly and Company, Indianapolis, IN, USA 1 Clinical Cooperation Unit Neuro-oncology, German Cancer 7 BioStat Solutions, Inc, Frederick, MD, USA Research Center, Heidelberg University Medical Center, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany 8 Department of Neuropathology, Charité – Universitätsmedizin 2 The Preston Robert Tisch Brain Tumor Center at Duke, Duke Berlin, corporate member of Freie Universität Berlin, University, Durham, NC, USA Humboldt-Universität zu Berlin, and Berlin Institute of Health, 3 Berlin, Germany Vall d’Hebron University Hospital and Institute of Oncology, Universitat Autònoma de Barcelona, Barcelona, Spain 9 Department of Investigational Cancer Therapeutics, Division of 4 H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, Cancer Medicine, U. T. M. D. Anderson Cancer Center, USA Houston, TX, USA Invest New Drugs (2020) 38:1570–1579 1571 Introduction (Phase 1b/2a) malignant glioma. Patients with newly di- agnosed and untreated intracranial GB including lower Glioblastoma (GB) is the most common and aggressive brain grade glioma, which evolved into GB were eligible. cancer representing approximately 15% of all primary brain Patients with moderate or severe cardiac disease were tumors, and about 55% of all gliomas [1]. The standard of care not eligible. Adequate hematologic, hepatic, and renal for patients with GB consists of maximal surgical resection function, and a performance status of ≤ 1 on the followed by radiotherapy (RTX) with concomitant and main- Eastern Cooperative Oncology Group (ECOG) scale tenance temozolomide (TMZ). This therapy results in were required. Concurrent use of stereotactic radiosur- progression-free survival (PFS) at 6 months of 53.9% and gery was not allowed. A biopsy or resection was re- median overall survival of 14.6 months [2]. Most of the quired no more than 6 weeks prior to treatment and an therapy-responsive patients will die within a period of 2 years MRI was required within 72 h after surgery; measurable and the 2- and 5-year overall survival rates are 27% and 9.8%, or assessable disease was not required. Patients were re- respectively [3]. quired to begin study treatment within 2–6 weeks after GB is characterized by persistent angiogenesis at the tumor surgery. site, and decreased peripheral immune responsiveness in pa- This study was conducted according to the principles of tients [4]. GB microenvironment is enriched in immunosup- good clinical practice, applicable laws and regulations, and pressive molecules such as transforming growth factor the Declaration of Helsinki. The protocol was approved by (TGF)-β that plays a specific role in cancer cell growth [5], each institution’s review board. This study was conducted in in addition to affecting immune cell response, and endothelial 9 centers in 3 countries. Between April 2011 and August cell and fibroblast differentiation [6, 7]. 2015, 101 patients entered the study but only 75 patients were TGF-β is a multifunctional cytokine that is involved in a enrolled and received at least one dose of galunisertib or TMZ variety of cell functions including cell proliferation, migration, (patients on therapy; Online Resource: Supplemental Fig. 1). survival, and death that influence tumor growth in advanced All patients provided written informed consent. This trial is forms of cancer [6, 7]. Upon binding to their ligands registered with ClinicalTrials.gov (NCT01220271). (TGF-β1, 2, and 3), the TGF-β kinase receptors are phosphor- ylated triggering phosphorylation of SMAD2 and SMAD3, Study design and formation of SMAD complexes [8, 9]. Galunisertib is an oral small molecule inhibitor of TGF-β In Phase 1b, two dose levels of galunisertib (2 cohorts: 160 mg/ kinase receptor type I (TGF-β RI/ALK5) [10] and selectively day or 300 mg/day) in combination with radiochemotherapy inhibits the serine/threonine activity of the receptor, thereby were studied to determine the dose for the Phase 2a portion of preventing the phosphorylation of downstream proteins, the study (Online Resource: Supplemental Fig. 2A). In Phase 2a, SMAD2 and SMAD3 [10]. The antitumor activity of eligible patients were enrolled, and randomized (3:1) to either galunisertib has been demonstrated in three different in vivo galunisertib at 300 mg/day plus radiochemotherapy, or to a con- tumor models; two breast cancer models, MX1 and 4 T1; and trol arm of radiochemotherapy (Online Resource: Supplemental a non-small cell lung cancer model, Calu6 and fibrosis Fig. 2A). Patients received galunisertib on an intermittent dose [11–13]. regimen of 14 days on/14 days off for a 28-day cycle (Online Based on the role of TGF-β in patients with malignant GB, Resource: Supplemental Fig. 2B). evidence of antitumor effects of TGF-β inhibitors such as galunisertib (including in a monotherapy study in glioblasto- Study treatment ma), and a favorable short- and long-term toxicity profile [14, 15], a multicenter Phase 1b/2a clinical trial was initiated to RTX consisted of 30 fractions at 1.8 to 2.0 Gy/dose (5 days a investigate the clinical benefit of combining galunisertib with week for 6 weeks) for a total dose up to 60 Gy (Online standard TMZ-based radiochemotherapy (TMZ/RTX) in pa- Resource: Supplemental Fig. 2B). Galunisertib was given tients with newly diagnosed malignant glioma. orally twice daily as 150 mg tablets for 14 days on/14 days off. TMZ was administered as recommended [2]. All patients received at least 6 cycles of therapy until disease progression, Methods death, or discontinuation due to adverse events (AEs), or other reasons. Patients Safety assessments Eligible male and female patients were 18 years and older with histologically proven, World Health Safety was evaluated on all patients (Phase 1b and 2a) who Organization Grade III (Phase 1b part only) and IV received at least one dose of galunisertib or TMZ. Safety 1572 Invest New Drugs (2020) 38:1570–1579 analyses included AE rates, laboratory and non-laboratory Statistical methods changes, physical examination and other safety observations including cardiac safety, such as echocardiography/Doppler, Patient disposition, demographic, safety, drug-related treat- chest CT scan, and cardiac plasma markers (brain natriuretic ment-emergent adverse events (TEAEs), and response data protein, Troponin I, Cystatin C and high sensitivity C-reactive were summarized using patient number, frequency counts, protein). or percentages as appropriate. The safety analysis was based on summaries of AEs reported in Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, and possi- Efficacy assessments bly drug related. All time-to-event variables were analyzed using the Primary objective of the Phase 1b study was to determine the Kaplan-Meier method with 90% confidence interval safe and tolerable Phase 2a dose of galunisertib in patients (CI). Univariate Cox models were used to evaluate results treated concomitantly with radiochemotherapy, and the phar- for potential prognostic markers by considering their im- macokinetics (PK) of galunisertib in combination with TMZ. pact on OS and PFS. Continuous markers were first con- Primary objective of the Phase 2a study was to confirm the verted to 2-level categorical variables by dichotomizing at tolerability, and evaluate the pharmacodynamic (PD) effect on the median and hazard ratios between treatment arms es- T-cells of galunisertib when combined with TMZ-based radio- timated for each level. chemotherapy in patients with GB, as measured by changes in The absolute cell number of Treg cells, CD4+, and CD8+ T response biomarkers and their relationship to clinical benefit cells from each patient were presented in profile plots together (overall survival [OS]). The secondary objectives were to with the geometric mean and 90% CI at baseline, Day 42 and evaluate time-to-event variables such as progression-free sur- Day 182 for each cell type. Pair-wise t-Tests were used to vival (PFS), time-to-treatment failure (TTF), time-to-tumor estimate the change from baseline to Day 42 for T cell subsets progression (TTP), duration-to-tumor response (DTR), over- in each arm. all response rate and clinical benefit rate. Galunisertib PK was also characterized (Online Resource: Supplemental Methods). Assessment of tumor response was based on Response Results Assessment in Neuro-Oncology (RANO) criteria [16] (Online Resource: Supplemental Methods). Phase 1b A Phase 1b study was performed to determine a safe and Pharmacodynamics of biomarkers tolerable Phase 2a dose of galunisertib in combination with radiochemotherapy in patients with GB (Online Tumor tissue and blood samples were collected at baseline Resource: Supplemental Fig. 1). PK analysis for and at specified times post-baseline. The baseline expression galunisertib was also completed at two dose levels of of tissue biomarkers including glial fibrillary acidic protein galunisertib (160 mg/day [n = 10] or 300 mg/day [n = 9]) (GFAP), Ki67, CD3, phospho-SMAD2 (pSMAD2), and given to Phase 1b eligible patients (Online Resource: isocitrate dehydrogenase 1 (IDH1) R132H was evaluated by Sup pl eme nt al Ta ble 2). The do se e sc al ati on o f immunohistochemistry staining and scoring method as de- galunisertib to 300 mg/day did not increase the toxicity scribed previously [17] (Online Resource: Supplemental profile, and overlapping toxicity was not observed when Methods). combined with radiochemotherapy (Online Resource: Patients’ hematology, and expression of lactate dehy- Supplemental Table 3). The Phase 1b PK data were con- drogenase (LDH), YKL-40, and serum S100β were deter- sistent with previous PK analyses of galunisertib (Online mined by clinical laboratory tests. Plasma TGF-β and Resource Supplemental Fig. 4A-C). Based on overall tox- MDC/CCL22 were measured by enzyme-linked immuno- icity and PK information from Phase 1b, 300 mg/day of sorbent assay (ELISA) (R&D systems), and multi-analyte galunisertib was selected for the Phase 2a part of the immunoassay panel (MAIP) of 47 analytes (Myriad/ study (Online Resource: Supplemental Results). RBM), respectively. Blood samples from patients were collected and prepared Patient dispositions, demographics, and baseline for flow cytometry by Quintiles laboratories (Durham, NC) to characteristics determine the expression of CD3+ T cell subsets, such as CD4+ and CD8+, and T regulatory cells defined as CD4 + A total of 59 patients from multiple centers were randomly CD25 + CD127-FoxP3+. Cell staining strategy is described in assigned to the Phase 2a study, and 56 patients received at Online Resource: Supplemental Methods. least one dose of galunisertib at 300 mg/day [14] Invest New Drugs (2020) 38:1570–1579 1573 Phase 2a 59 patients randomized 56 patients enrolled and received ≥1 dose Galunisertib + TMZ/RTX TMZ/RTX (N=40) (N=16) ● Reason for treatment discontinuation: - Progressive disease n=30 ● Reason for treatment discontinuation: - Adverse event n=1 - Progressive disease n=7 o Grade 4 Pneumonitis - Sponsor decision n=1 - Subject decision n=4 - Subject decision n=3 - Physician decision n=4 - Physician decision n=4 - Consent withdrawn n=1 - Consent withdrawn n=1 ● Reason for study discontinuation: ● Reason for study discontinuation: - Subject decision n=2 - Death n=11 - Death n=31 o Study disease n=9 o Study disease n=26 o Adverse eventa n=1 o Adverse eventsa n=2 o Not reported n=1 o Not reported n=3 ● Treatment cycles received per patient before discontinuation: ● Treatment cycles received per patient before discontinuation: - Galunisertib: Median 6.5 cycles Range 2-23 cycles - TMZ: Median 7 cycles, - TMZ: Median 6 cycles, Range 2-14 cycles Range 2-15 cycles ● Anti-cancer therapies after study drug discontinuation, n (%) ● Anti-cancer therapies after study drug discontinuation, n (%) - Patients receiving ≥ 1 therapy, n=6 (37.5) - Patients receiving ≥ 1 therapy, n=22 (55) - Antineoplastic agents: - Antineoplastic agents: o Bevacizumab n=0 (0.0) o Bevacizumab n=10 (25) o TMZ n=2 (12.5) o TMZ n=6 (15) Fig. 1 Patient dispositions from treatment. Abbreviations: TMZ = temozolomide; RTX = radiation. aAEs not-related to study treatment as AEs happened >30 days after discontinuation of study treatment. concomitantly with TMZ-based radiochemotherapy (n = 40) Efficacy of treatment or TMZ-based radiochemotherapy (n = 16; Fig. 1). Among the Phase 2a patients, 38/56 (67.9%) had received complete Overall survival was 18.2 months (95% CI: 13.4, 20.6 months) or partial surgery as the only prior treatment for their disease; in the galunisertib plus radiochemotherapy arm compared to 26/40 (65%) patients who had prior surgery were treated with 17.9 months (95% CI: 10.7, 24 months) in the radiochemo- galunisertib plus radiochemotherapy; and 12/16 (75%) pa- therapy arm (Fig. 2a-c). Galunisertib plus radiochemotherapy, tients were treated in the radiochemotherapy alone arm and radiochemotherapy alone had a censoring rate of 22.5% (Table 1). and 31.3%, respectively. The galunisertib plus radiochemotherapy arm showed a median PFS of 7.6 months (95% CI: 6.1, 10.4 months), and Treatment discontinuation the probability that PFS at 12 and 24 months, was 25% (90% CI: 14, 37%) and 5% (90% CI: 1, 14%), respectively (Fig. 2b The major reason for treatment discontinuation was progres- and c). In comparison, the radiochemotherapy arm had a me- sive disease (37/56 [66.1%] (Fig. 1). By the end of the study, dian PFS of 11.5 months (95% CI: 5.4, 15.9 months) (Fig. 2b 42/56 (75%) patients from the two treatment arms had died and c). (galunisertib plus radiochemotherapy, 31/40 [77.5%]; radio- Assessment of tumor response showed that 3 out of chemotherapy, 11/16 [68.8%]). Of those who died, 35/56 40 patients (7.5% [90% CI: 2.1, 18.3%]) from [62.5%] were due to disease progression, 3/56 [5.4%] were galunisertib plus radiochemotherapy arm had a complete due to AEs that started 30 days after study treatment discon- response compared to none in radiochemotherapy arm tinuation and cause of death was not reported for 4/56 [7.1%]). (Online Resource: Supplemental Table 1). Therefore, Among the 56 patients enrolled in the Phase 2a clinical trial, the overall disease control rate in the galunisertib plus 28 (50%) patients moved to other anti-cancer therapies (main- radiochemotherapy arm was higher than in the radioche- ly bevacizumab and TMZ) after treatment discontinuation due motherapy arm (80% [90% CI: 66.8, 89.6%] vs 56.3% to disease progression (Fig. 1). [90% CI: 33.3, 77.3]). 1574 Invest New Drugs (2020) 38:1570–1579 Table 1 Patient demographics and baseline characteristics Characteristics (Phase 2a) Gal + TMZ/RTX TMZ/RTX Total N = 40 N = 16 N = 56 Gender, Male 22 (55.0) 11 (68.8) 33 (58.9) Age, years, mean, (SD) 58.7 (8.9) 57.8 (11.6) 58.4 (9.7) Race, White 39 (97.5) 15 (93.8) 54 (96.4) ECOG PS 0 14 (35.0) 8 (50.0) 22 (39.3) 1 26 (65.0) 7 (43.8) 33 (58.9) Missing 0 (0.0) 1 (6.3) 1 (1.8) Basis of initial pathological diagnosis Histopathological 39 (97.5) 16 (100) 55 (98.2) Missing 1 (2.5) 0 (0.0) 1 (1.8) Study entry pathological diagnosis Glioblastoma 39 (97.5) 16 (100) 55 (98.2) Glioma, oligodendroglioma 1 (2.5) 0 (0.0) 1 (1.8) Prior treatment Surgery 26 (65.0) 12 (75.0) 38 (67.9) Partial 14 (35.0) 6 (50.0) 20 (35.7) Complete 12 (30.0) 6 (50.0) 18 (32.1) To note: data given as No. (%) unless otherwise indicated Gal galunisertib, TMZ temozolomide, RTX radiation, SD standard deviation, ECOG PS Eastern Cooperative Oncology Group performance status Safety profile (AEs) 3B). The subgroup most favoring galunisertib plus radioche- motherapy treatment compared to control was a high ratio of Possible all grade drug-related TEAEs (determined by CD4/CD8, however it was not statistically significant. CTCAE grade) occurring at a frequency of ≥ 10% of patients Next, we evaluated the T cell counts in both treatment arms are described in Table 2. The most common (≥ 20%) all grade every 2 weeks for 24 weeks (Fig. 3a). Variability within and laboratory events in galunisertib plus radiochemotherapy arm between patients was observed, but when the geometric means were platelet count decreased (45%), lymphocyte count de- were assessed at baseline, Day 42 (end of radiation therapy), and creased (32.5%), and white blood cells decreased (30%). The Day 182 (adjuvant phase), it was apparent that CD4 and CD8 percentage of grade 3–4 drug-related TEAEs was higher in lymphocytes in galunisertib plus radiochemotherapy arm were patients treated with galunisertib plus radiochemotherapy numerically decreased during radiochemotherapy (i.e., Day 42) compared to radiochemotherapy alone. Grade 3–4 most com- as previously reported for CD4 T cells [18]. A pair-wise T-test mon drug-related TEAEs (≥ 10%) were decreases in platelet, analysis comparing the geometric mean of CD4, CD8, and Treg lymphocyte, and white blood cell count. The most commonly cells at baseline and Day 42 showed a significant decrease in reported (≥ 20%) all grade non-laboratory events in the these cell types in the galunisertib plus radiochemotherapy arm galunisertib plus radiochemotherapy arm were fatigue (Baseline vs Day 42: 545.0 vs 260.2 [CD4]; 227.2 vs 131.8 (62.5%), nausea (52.5%), constipation (40%), alopecia [CD8]; 19.8 vs 12.1 [Treg]; Fig. 3b). After radiation, the cell (40%), vomiting (30%), and appetite decreased (20%). counts stayed steady or slightly recovered over time when galunisertib and TMZ were given as an adjuvant treatment. Correlation between biomarkers and clinical benefit After 200 days of galunisertib plus radiochemotherapy treatment, approximately 22 weeks after radiation (≈ Cycle 8) and during Describing and comparing the effect of galunisertib in combi- adjuvant treatment with TMZ, we observed two clusters of pa- nation with TMZ-based radiochemotherapy on major subsets tients, one with a high number of CD8 T cells (11/16 patients), of circulating immune cells, particularly Tregs (CD4 + the other with a low cell number (5/16 patients) by a cutoff of CD25 + CD127-FoxP3+ T cells), was one of the main trans- around 120 cells/μl (Fig. 3a). This may suggest that galunisertib lational objectives of this study (Online Resource: treatment may provide some protective effect on CD8 T cell Supplemental Fig. 3). The analysis of OS/PFS across baseline population in a subset of patients. biomarkers showed no association between baseline Tregs Previous studies identified IDH1 R132H mutation as a posi- and OS/PFS (Online Resource: Supplemental Fig. 3A and tive prognostic marker of GB that occurs with a prevalence of Invest New Drugs (2020) 38:1570–1579 1575 a 1.0 Gal + TMZ/RTX TMZ/RTX b 1.0 Gal + TMZ/RTX TMZ/RTX 95% CI 95% CI 95% CI 95% CI 0.9 0.9 0.8 0.8 PFS (probability) OS (probability) 0.7 0.7 0.6 0.6 median median 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 0 4 8 12 16 20 24 28 32 36 0 4 8 12 16 20 24 28 32 36 Time (months) Time (months) Number 40 39 36 27 22 14 9 5 2 0 Number 40 35 18 9 4 3 2 0 0 0 of patients of patients at risk 16 15 14 12 9 6 4 2 1 0 at risk 16 14 10 7 4 3 2 1 1 0 c Median, m 12-month (%) 24-month (%) Censoring Hazard ratio Arms (95% CI) (90% CI) (90% CI) Rate (%) (90% CI) Gal + TMZ/RTX 18.2 (13.4, 20.6) 74 (59, 83) 27 (16, 39) 22.5 OS TMZ/RTX 17.9 (10.7, 24.0) 80 (56, 92) 33 (15, 53) 31.3 1.2 (0.7, 2.1) Gal + TMZ/RTX 7.6 (6.1, 10.4) 25 (14, 37) 5 (1, 14) 7.5 PFS TMZ/RTX 11.5 (5.4, 15.9) 47 (25, 66) 13 (3, 31) 18.8 1.8 (1.0, 3.1) Gal + TMZ/RTX 7.1 (5.5, 8.9) NR NR 22.5 TTF TMZ/RTX 8.8 (5.1, NR) NR NR 56.3 1.3 (0.6, 2.6) Gal + TMZ/RTX 7.2 (6.1, 10.3) 24 (13, 37) 5 (1, 15) 22.5 TTP TMZ/RTX 11.5 (6.8, 22.5) 43 (20, 64) 21 (5, 44) 43.8 2.0 (1.1, 3.9) Gal + TMZ/RTX 3.7 (1.9, 7.6) NR NR NR DTR TMZ/RTX NR NR NR NR 1.7 (0.3, 11.2) Fig. 2 Summary of treatment responses. Kaplan-Meier estimates of OS free survival; TFF = time-to-treatment failure; TTP = time-to-tumor (a), and PFS (b). Summary of OS, PFS, TTF, TTP, and DTR (c). progression; DTR = duration of tumor response; Gal = galunisertib; Abbreviations: m = months; OS = overall survival; PFS = progression- TMZ = temozolomide; RTX = radiation; NR = Not reported almost 85% in secondary GB and approximately 5% in primary studies done during Phase 2a showed that the plasma levels of GB [19]. In the present study, the IDH1 R132H mutation was galunisertib were not altered when combined with TMZ and found on 6 of the 78 (8%) Phase 1b/2a-tested patients and all 6 radiation (Online Resource: Supplemental Fig. 4) and achieved were treated with galunisertib plus radiochemotherapy, 4 in the the targeted biologically effective dose level. Phase 1b Cohort (data not shown) and 2 in the Phase 2a Cohort While both treatments showed comparable results for median (Online Resource: Supplemental Table 4). OS (18.2 vs 17.9 months), the galunisertib plus radiochemother- Prior to therapy, the expression of the following known apy group had a shorter estimated PFS than the radiochemother- tumor tissue biomarkers CD3, Ki67, GFAP, and pSMAD2 apy group (7.6 vs 11.5 months). This difference might be ex- were assessed (Online Resource: Supplemental Table 4). No plained by the small number of patients in both arms or the earlier association between these factors and clinical outcome were withdrawal of patients from galunisertib plus radiochemotherapy identified (data not shown). In addition, the PD analysis con- arm. For example, 55% of patients from the experimental arm ducted to determine the effect of galunisertib plus radioche- were moved to subsequent therapies, versus 37.5% of patients motherapy treatment on plasma markers (LDH, YKL-40, from control received other therapies (Fig. 1). S100β, TGF-β and MDC/CCL22) did not find any associa- The overall safety data across treatment arms was similar; tion between the treatment and biomarkers (data not shown). however, the frequency of grade 3–4 toxicities was higher in the galunisertib plus radiochemotherapy arm. There was a severe case of myeloablative marrow aplasia during the first Discussion cycle of treatment in a Phase 1a patient; this finding is more likely related to the known side effect of TMZ and radiation We here report the efficacy, safety, and PD of galunisertib com- than to galunisertib treatment [20]. Galunisertib was not asso- bined with radiochemotherapy in newly diagnosed malignant ciated with bone marrow side effects in preclinical toxicology glioma. The overall toxicity and PK results from the Phase 1b studies evaluating galunisertib in human bone marrow assays study (Online Resource: Supplemental Results) were used to or in other combination studies with chemotherapeutic agents determine the recommended Phase 2a dose. Additionally, PK [14]. 1576 Invest New Drugs (2020) 38:1570–1579 Table 2 Drug-related TEAEs by CTC grade (≥10%) Gal + TMZ/RTX (N = 40) TMZ/RTX (N = 16) Grade 1–2 Grade ≥3 All Grade Grade 1–2 Grade ≥3 All Grade Laboratory Event Platelet count decreased 12 (30.0) 6 (15.0) 18 (45) 3 (18.8) 1 (6.3) 4 (25.1) Lymphocyte count decreased 5 (12.5) 8 (20.0) 13 (32.5) 2 (12.5) 1 (6.3) 3 (18.8) White blood cells decreased 8 (20.0) 4 (10.0) 12 (30) 1 (6.3) 0 (0.0) 1 (6.3) ALT increased 4 (10.0) 3 (7.5) 7 (17.5) 2 (12.5) 0 (0.0) 2 (12.5) Neutrophil count decreased 5 (12.5) 2 (5.0) 7 (17.5) 2 (12.5) 0 (0.0) 2 (12.5) Anaemia 6 (15.0) 0 (0.0) 6 (15) 0 (0.0) 0 (0.0) 0 (0.0) Non-laboratory Event Fatigue 24 (60.0) 1 (2.5) 25 (62.5) 6 (37.5) 0 (0.0) 6 (37.5) Nausea 21 (52.5) 0 (0.0) 21 (52.5) 8 (50.0) 1 (6.3) 9 (56.3) Constipation 16 (40.0) 0 (0.0) 16 (40) 4 (25.0) 0 (0.0) 4 (25.0) Alopecia 16 (40.0) 0 (0.0) 16 (40) 3 (18.8) 0 (0.0) 3 (18.8) Vomiting 11 (27.5) 1 (2.5) 12 (30) 5 (31.3) 0 (0.0) 5 (31.3) Decreased Appetite 8 (20.0) 0 (0.0) 8 (20) 2 (12.5) 0 (0.0) 2 (12.5) Headache 6 (15.0) 0 (0.0) 6 (15) 2 (12.5) 0 (0.0) 2 (12.5) Dyspepsia 5 (12.5) 0 (0.0) 5 (12.5) 1 (6.3) 0 (0.0) 1 (6.3) Weight decreased 5 (12.5) 0 (0.0) 5 (12.5) 1 (6.3) 0 (0.0) 1 (6.3) To note: data given as No. (%) unless otherwise indicated ALT alanine aminotransferase, Gal galunisertib, TMZ temozolomide, RTX radiation, TEAE treatment-emergent adverse events In addition, because cardiovascular toxicities are associated and mean PFS of 14.4 months, and the 11 patients with a higher with small molecule inhibitors of TGF-β signaling in preclin- number of CD8+ cells had a mean OS of 19.4 and mean PFS of ical toxicology studies [21], cardiac toxicity was monitored in 13.1 months. However, these observations need further confir- all patients. Galunisertib treatment did not show any clinically mation in larger cohorts of patients. significant cardiac safety concerns, which are consistent with Furthermore, we found no association between OS and previous reports for a TGF-β small molecule inhibitor [22]. MDC/CCL22 contrary to reports for second line treatment Biomarker studies did not find any correlation between base- of GB patients [26]. It is possible that baseline levels of line T cell subsets (including Tregs) and OS or PFS (Online MDC/CCL22 are different between first and second line pa- Resource: Supplemental Fig. 3A and 3B). As reported by others, tients. Additionally, we found no association between the CD4+, CD8+, and Treg cells count at 10 weeks post radio- pSMAD2 levels in tumor tissue and OS. The presence of chemotherapy treatment were numerically decreased [23, 24]. CD3+ T cells in tumor tissue was not associated with OS The longitudinal analysis shows early decrease of CD4+ and changes. These findings are different from those reported for CD8+ T cell counts during radiation, followed by a steady phase the second line patients treated with galunisertib [17]. or a slight recovery in these cells over time within the galunisertib TGF-β is a major driver of glioma progression, via its role plus radiochemotherapy arm, while the pattern of CD4+, CD8+, in tumor cell proliferation and invasion, angiogenesis, and and Treg cell counts were steady over time in the radiochemo- immune suppression within the tumor microenvironment therapy arm during both radiation and post-radiation phases. In [27]. Blocking TGF-β signaling by inhibition of its receptor other diseases, such as lung cancer, transient decreases in CD8+ TGF-β RI is one strategy for abrogating its pro-tumorigenic T cells followed by an increase is associated with better OS [25]. effects. Galunisertib is one of the only drugs in development Hence, the relevance of our observation needs further examina- designed to specifically target TGF-β RI; it is furthest along tion in order to decide whether such a response would be also the clinical trial pipeline [28], not only in the setting of recur- expected in GB patients. rent glioma/GB (in combination with TMZ/RTX in the current An exploratory analysis was performed to examine whether study and in combination with lomustine in [15, 26]), but also any two clusters of patients with respect to OS and PFS emerged for other solid tumors. Galunisertib is currently being evalu- after 200 days of galunisertib plus radiochemotherapy treatment ated in Phase 1/2 or Phase 2 studies in combination with (Fig. 3a). This limited analysis set showed that the 5 patients with immune checkpoint inhibitors [29, 30], sorafenib [31], and a low number of CD8+ T cells had a mean OS of 25.5 months gemcitabine [32]. In a Phase 2 study in patients with Invest New Drugs (2020) 38:1570–1579 1577 a Gal + TMZ/RTX TMZ/RTX Tregs (cells/PL) Patients 150 Geometric mean (Gal + TMZ/RTX) Geometric mean (TMZ/RTX) 100 50 0 CD4+ (cells/PL) 2000 1000 0 1250 CD8+ (cells/PL) 1000 750 500 250 0 0 100 200 300 0 100 200 300 Relative nominal day since first dose b T cell subsets Arms Baseline, Day42 Day182 P-value GM [90% CI], n GM [90% CI], n GM [90% CI], n (Baseline vs Day42) Gal + TMZ/RTX 19.8 (16.8, 23.3), 38 12.1 (10.1, 14.4), 35 19.3 (15.2, 24.5), 17 ** 0.0013 Tregs (cells/PL) TMZ/RTX 14.7 (9.2, 23.4), 14 18.4 (15.2, 22.3), 12 22.2 (16.8, 29.5), 7 0.4934 Gal + TMZ/RTX 545.0 (470.8, 630.9), 38 260.2 (221.9, 305.2), 35 279.5 (226.8, 344.5), 17 *** <0.0001 CD4+ (cells/PL) TMZ/RTX 441.3 (299.7, 649.8), 14 389.8 (312.7, 486.0), 12 287.7 (220.7, 375.0), 7 0.6654 Gal + TMZ/RTX 227.2 (189.2, 272.9), 38 131.8 (101.8, 170.7), 35 182.9 (128.5, 260.2), 17 ** 0.0055 CD8+ (cells/PL) TMZ/RTX 294.5 (208.7, 415.4), 14 264.4 (213.3, 327.6), 12 293.3 (212.4, 405.0), 7 0.6779 Fig. 3 Characterization of the changes in the major T cell subsets in Phase beginning of the adjuvant phase. b Geometric mean, with 90% 2a. a Absolute numbers of CD4 + CD25 + CD127-FoxP3+ T (Tregs), confidence interval, at baseline, Day 42, and Day 182. The p value CD4+, and CD8+ T cells are reported over time from the first dose of from Pair-wise t-Test compares baseline and Day 42 within each arm. treatment. Each gray line represents a patient. Solid and dashed lines Note: Day42 = End of Radiation Phase, Cycle 2 Day 14; Day182 = Cycle connect the geometric mean (error bars, 90% CI) at baseline, Day 42 7 day 14; P value = Pair-wise t-Test for Comparing Baseline and Day42. (end of radiation phase), and Day 182 (adjuvant phase). Vertical dotted Abbreviations: Gal = galunisertib; TMZ = temozolomide; RTX = line indicates the end of the chemoradiotherapy treatment and the radiation pancreatic cancer, the first line treatment of galunisertib in contrast to these small molecule approaches, large molecule combination with gemcitabine resulted in an OS benefit of development has had advances, including M7824 (bintrafusp 8.9 months compared to 7.1 months for patients receiving alfa), which is currently being evaluated in registration stud- gemcitabine alone (hazard ratio [HR], 0.79; 95% CI, 0.59– ies, including for NCSLC [37]. 1.09) [32]. In HCC patients with elevated alpha-fetoprotein In conclusion, the combination of galunisertib with stan- prior to treatment and who had previously progressed on so- dard radiochemotherapy did not accentuate the toxicity profile rafenib or were considered not eligible to receive sorafenib, of the radiochemotherapy. Even though survival probability galunisertib monotherapy achieved a median OS of was unchanged between the two treatments and PFS was re- 7.3 months (95% CI, 4.9–10.5). OS was longer for those pa- duced in the galunisertib plus radiochemotherapy arm, the tients who showed reduced alpha-fetoprotein (>20% from disease control rate was higher in the galunisertib plus radio- baseline) compared to non-responders (21.5 months vs chemotherapy treatment when compared to radiochemothera- 6.8 months) [33]. While these signals were encouraging, the py treatment alone. Due to new R&D priorities, Eli Lilly sponsor discontinued future clinical development for discontinued the development of galunisertib in 2017 [34]. galunisertib in mid-2017 [34]. Other TGF-β RI inhibitory drugs in early clinical development (Phase 1) include Acknowledgements The authors thank Loretta Wilcox for contributions to this manuscript, and Annie-Carole Trampont, PhD, Eli Lilly and Company, LY3200882 [35] and vactosertib [36], however there are no for her assistance in writing this manuscript. We thank patients and their published reports of efficacy of these compounds as of yet. In 1578 Invest New Drugs (2020) 38:1570–1579 families who participated in this trial, and all site staff and investigators at the References institutions, and the trial personnel at Eli Lilly and Company. 1. Glioblastoma Facts and Figures. National Brain Tumor Society. https:// Authorship All authors have contributed to the conception and the de- braintumor.org/take-action/about-gbm/. Accessed 17 Jan 2020 sign of the work, the data acquisition, the analysis and/or the interpreta- tion of the data, in addition to drafting and/or the critical revision of the 2. 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