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Supplementary methods of tumor xenograft studies and light microscopic evaluation of tumor xenografts

AMG 337 KINOME scan binding data

Supplementary Figure 1: Assessment of kidney cancer cell lines A498, 786-O, and A704 via flow cytometry revealed surface expression of P-gp in all lines; Supplementary Figure 2: 786-O and A704 cells were transduced with lentiviral FR1, and flow cytometric analysis shows increased surface FR1 expression; Supplementary Figure 3: NCI/ADR-RES or NCI/ADR-RES-FR cells with known FR levels were treated with increasing concentrations (10-9 M to 10-5 M) of vintafolide for 2 h; Supplementary Figure 4: Ovarian tumors positive for P-gp tend to express lower levels of FR; Supplementary Table 1: P-gp inhibitor elacridar reverses P-gp-mediated DAVLBH and vintafolide resistance; Supplementary Table 2: Functional multi-drug transporter efflux assay.

Tumor cell line profiling data

The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568–79. ©2016 AACR.

High-level focal amplification of MET is associated with sensitivity to AMG 337

Figure S1. Kinase interaction map for AMG 337; Figure S2. In a large unbiased cancer cell line viability screen only MET-amplified cell lines were sensitive to treatment with an analogue of AMG 337 (Compound 5); Figure S3: AMG 337 inhibits the phosphorylation of MET and but not its downstream effectors in MET-amplified, KRAS mutant NSCLC cell line NCI-H1573; Figure S4. Cell lines harboring MET FISH scores >3 exhibited sensitivity to AMG 337. MET FISH analysis was performed on a subset of cancer cell lines exhibiting elevated MET gene copy number; Figure S5. Selective inhibition of MET exhibits partial effects on the viability of U-87 MG glioblastoma cells harboring an HGF/MET autocrine loop; Figure S6. Increases in MET gene number correlate with high levels of total MET protein; Figure S7. AMG 337 inhibits Gab-1 phosphorylation in a concentration dependent manner in the TPR-MET mouse tumor model.

Legends for Supplementary Figures 1-4 and Supplementary Tables 1 & 2

Supplementary Table S3 shows inconsistencies between confirmed best overall responses.

Supplementary Figure S2 shows the Kaplan-Meier plots of investigator-assessed progression-free survival.

Purpose:Patients with advanced gastric/gastroesophageal junction cancer (GC/GEJC) have limited treatment options after first-line therapy. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGFβRII receptor (a TGFβ “trap”) fused to a human IgG1 antibody against programmed death ligand 1 (PD-L1), potentially offering a new treatment approach for these patients. We report results for bintrafusp alfa in GC/GEJC.Patients and Methods:Asian patients with recurrent GC/GEJC for whom standard therapy does not exist or for whom standard therapy has failed enrolled in this expansion cohort of an ongoing phase I trial and received bintrafusp alfa 1,200 mg once every 2 weeks until disease progression, unacceptable toxicity, or withdrawal. The primary objective was to assess safety/tolerability.Results:By July 23, 2018, 31 heavily pretreated patients received bintrafusp alfa for a median of 10.1 weeks; 3 patients remained on treatment. Six patients (19%) experienced grade 3 treatment-related adverse events (AE); no grade 4 events occurred. One on-treatment death occurred (sudden death); rupture of a preexisting thoracic aortic aneurysm was the suspected cause. Ten patients (32%) had immune-related AEs. The confirmed objective response rate per independent review committee was 16%; disease control rate was 26%. Median duration of response was 8.7 months (range, 2.4–12.4+). Responses occurred irrespective of PD-L1 expression or microsatellite instability status and appeared to correlate with high tumor TGFB1 levels.Conclusions:In this first evaluation in Asian patients with heavily pretreated advanced GC/GEJC, bintrafusp alfa demonstrated a manageable safety profile and clinical activity.

Supplementary Figure 2 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Supplementary Methods, Figures 1-5 from Preclinical Evaluation of AMG 900, a Novel Potent and Highly Selective Pan-Aurora Kinase Inhibitor with Activity in Taxane-Resistant Tumor Cell Lines

Supplementary Figure 1 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

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XLS file 3927K, STK33 enzymatic activity and cellular activities for molecules a-l from Figure 6E

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Recepteur d'origine nantais (RON) is a receptor tyrosine kinase closely related to c-Met. Both receptors are involved in cell proliferation, migration, and invasion, and there is evidence that both are deregulated in cancer. Receptor overexpression has been most frequently described, but other mechanisms can lead to the oncogenic activation of RON and c-Met. They include activating mutations or gene amplification for c-Met and constitutively active splicing variants for RON. We identified a novel inhibitor of RON and c-Met, compound I, and characterized its in vitro and in vivo activities. Compound I selectively and potently inhibited the kinase activity of RON and c-Met with IC50s of 9 and 4 nmol/L, respectively. Compound I inhibited hepatocyte growth factor–mediated and macrophage-stimulating protein–mediated signaling and cell migration in a dose-dependent manner. Compound I was tested in vivo in xenograft models that either were dependent on c-Met or expressed a constitutively active form of RON (RONΔ160 in HT-29). Compound I caused complete tumor growth inhibition in NIH3T3 TPR-Met and U-87 MG xenografts but showed only partial inhibition in HT-29 xenografts. The effect of compound I in HT-29 xenografts is consistent with the expression of the activating b-Raf V600E mutation, which activates the mitogen-activated protein kinase pathway downstream of RON. Importantly, tumor growth inhibition correlated with the inhibition of c-Met–dependent and RON-dependent signaling in tumors. Taken together, our results suggest that a small-molecule dual inhibitor of RON/c-Met has the potential to inhibit tumor growth and could therefore be useful for the treatment of patients with cancers where RON and/or c-Met are activated. [Cancer Res 2008;68(16):6680–7]

Supplementary Figure Legends 1-3 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

XLS file - 175K, Effects of 515 small molecules on STK33 enzymatic activity vs. phosphorylation of RPS6 in cell lines

Supplementary Figure 3 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

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Identification of low-dose, low-molecular-weight, drug-like inhibitors of protein-protein interactions (PPIs) is a challenging area of research. Despite the challenges, the therapeutic potential of PPI inhibition has driven significant efforts toward this goal. Adding to recent success in this area, we describe herein our efforts to optimize a novel purine carboxylic acid-derived inhibitor of the HDM2-p53 PPI into a series of low-projected dose inhibitors with overall favorable pharmacokinetic and physical properties. Ultimately, a strategy focused on leveraging known binding hot spots coupled with biostructural information to guide the design of conformationally constrained analogs and a focus on efficiency metrics led to the discovery of MK-4688 (compound 56), a highly potent, selective, and low-molecular-weight inhibitor suitable for clinical investigation.

We report the clinical activity, safety, and identification of a predictive biomarker for bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGFβRII (a TGF-β "trap") fused to a human IgG1 mAb blocking PD-L1, in patients with advanced triple-negative breast cancer (TNBC).In this expansion cohort of a global phase 1 study, patients with pretreated, advanced TNBC received bintrafusp alfa 1200 mg every 2 weeks intravenously until disease progression, unacceptable toxicity, or withdrawal. The primary objective was confirmed best overall response by RECIST 1.1 assessed per independent review committee (IRC).As of May 15, 2020, a total of 33 patients had received bintrafusp alfa, for a median of 6.0 (range, 2.0-48.1) weeks. The objective response rate was 9.1% (95% CI, 1.9%-24.3%) by IRC and investigator assessment. The median progression-free survival per IRC was 1.3 (95% CI, 1.2-1.4) months, and median overall survival was 7.7 (95% CI, 2.1-10.9) months. Twenty-five patients (75.8%) experienced treatment-related adverse events (TRAEs). Grade 3 TRAEs occurred in 5 patients (15.2%); no patients had a grade 4 TRAE. There was 1 treatment-related death (dyspnea, hemolysis, and thrombocytopenia in a patient with extensive disease at trial entry). Responses occurred independently of PD-L1 expression, and tumor RNAseq data identifiedBintrafusp alfa showed clinical activity and manageable safety in patients with heavily pretreated advanced TNBC.NCT02517398.

Adenocarcinoma esofàgic; Bintrafusp Alfa Adenocarcinoma Esofágico; Bintrafusp Alfa Esophageal Adenocarcinoma; Bintrafusp Alfa Background Esophageal adenocarcinoma patients have limited treatment options. TGF-β can be upregulated in esophageal adenocarcinoma, and blocking this pathway may enhance clinical response to PD-(L)1 inhibitors. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. Objective The objective of this study was to investigate the efficacy and safety of bintrafusp alfa in patients with advanced, post-platinum esophageal adenocarcinoma, unselected for PD-L1 expression. Patients and Methods In this phase 1 study, patients with post-platinum, PD-L1–unselected esophageal adenocarcinoma received bintrafusp alfa 1200 mg every 2 weeks until disease progression, unacceptable toxicity, or withdrawal. The primary endpoint was confirmed best overall response per RECIST 1.1 by independent review committee (IRC). Results By the database cutoff of 24 August 2018, 30 patients (80.0% had two or more prior anticancer regimens) received bintrafusp alfa for a median of 6.1 weeks. The confirmed objective response rate (ORR) per IRC was 20.0% (95% CI 7.7–38.6); responses lasted 1.3–8.3 months. Most responses (83.3%) occurred in tumors with an immune-excluded phenotype. Investigator-assessed confirmed ORR was 13.3% (95% CI 3.8–30.7). Nineteen patients (63.3%) had treatment-related adverse events: seven patients (23.3%) had grade 3 events; no grade 4 events or treatment-related deaths occurred. Conclusions Bintrafusp alfa showed signs of clinical efficacy with a manageable safety profile in patients with heavily pretreated, advanced esophageal adenocarcinoma. This trial was funded by Merck KGaA, Darmstadt, Germany, and is part of an alliance between Merck KGaA and GlaxoSmithKline. Merck KGaA provided the study drug and worked with investigators on the trial design and plan, collection and analysis of data, and interpretation of results. Funding for a professional medical writer with access to the data was provided by Merck KGaA and GlaxoSmithKline.

Background Patients with esophageal squamous cell carcinoma (SCC) have limited treatment options. Blocking transforming growth factor-β (TGFβ), which can be overexpressed in these tumors, may enhance responses to programmed cell death protein 1/programmed death-ligand 1 [PD-(L)1] inhibitors. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGFβ receptor II (TGFβRII) (a TGFβ “trap”) fused to a human IgG1 monoclonal antibody blocking PD-L1. Objective The objective of this study was to investigate the safety and efficacy of bintrafusp alfa in Asian patients with pretreated, PD-L1–unselected esophageal SCC. Patients and Methods In a phase 1 study, Asian patients with pretreated esophageal SCC received bintrafusp alfa 1200 mg every 2 weeks until disease progression, unacceptable toxicity, or withdrawal. The primary endpoint was safety/tolerability with a goal of exploring clinical activity. Results By the database cutoff of August 24, 2018, 30 patients (76.7% had two or more prior anticancer regimens) received bintrafusp alfa for a median of 6.1 weeks; two remained on treatment. Nineteen patients (63.3%) had treatment-related adverse events, seven (23.3%) with grade 3/4 events, and there were no treatment-related deaths. The confirmed objective response rate (ORR) per independent review was 10.0% (95% confidence interval [CI] 2.1–26.5); responses lasted 2.8–8.3 + months. All responses occurred in immune-excluded tumors. Investigator-assessed confirmed ORR was 20.0% (95% CI 7.7–38.6). Median overall survival was 11.9 months (95% CI 5.7–not reached). Conclusions Bintrafusp alfa demonstrated a manageable safety profile and efficacy in Asian patients with pretreated esophageal SCC. Clinical Trials Registration NCT02699515. Supplementary Information The online version contains supplementary material available at 10.1007/s11523-021-00810-9.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Identification of biomarkers that are predictive of response to investigational therapies will help identify patients that are more likely to clinically benefit from treatment. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In this expansion cohort of a global, phase 1, open-label trial (NCT02517398), an extensive tumor biomarker analysis was conducted to identify potential markers correlating with efficacy in patients with advanced TNBC treated with bintrafusp alfa. Methods: Patients received infusions of bintrafusp alfa 1200 mg every 2 weeks until confirmed progression, unacceptable toxicity, or trial withdrawal. Tumor archival material was collected from each patient for biomarker analysis. All biomarker analyses were exploratory. PD-L1 expression and immune phenotype were determined by immunohistochemistry. Tumor samples were also processed for RNA sequencing (RNAseq) and resulting data were analyzed to identify potential associations between gene expression and efficacy of bintrafusp alfa treatment. Results: As of August 24, 2018, 33 patients with heavily pretreated TNBC were treated with bintrafusp alfa (54.5% of patients received ≥4 prior regimens). The objective response rate was 9.1% (n=3) and the disease control rate was 15.2% in a total of 5 patients. PD-L1 expression levels in tumor cells and in the tumor microenvironment were generally low in this cohort with the majority of patients having 0% PD-L1+ tumor cells and/or ≤10% PD-L1+ immune cells in their tumor microenvironment. Moreover, several patients had an immune desert phenotype. PD-L1 expression was not associated with response to treatment. Importantly, exploratory analysis of RNAseq data (n=26 passing quality control) identified differential expression of the high mobility group AT-hook 2 (HMGA2) gene with 32.0-fold higher expression (as computed by DESeq2; q=2.23e−13) in patients who experienced disease control (response or stable disease) compared to those who had progressive disease. HMGA2 is a transcriptional regulator whose expression is upregulated by TGF-β signaling. Furthermore, HMGA2 is known to be an important factor in mediating TGF-β-induced epithelial-mesenchymal transition. In The Cancer Genome Atlas dataset, approximately 12% of subjects annotated as TNBC have high expression of HMGA2. Clinical efficacy and safety data for this expansion cohort are presented in a separate abstract. Conclusions: We identified high expression of HMGA2 as a potential predictive biomarker of response in TNBC patients treated with bintrafusp alfa. HMGA2 assay development is on-going to confirm this finding in a future clinical trial. Citation Format: George Locke, Yue Zhang, Laureen S Ojalvo, Christoph Helwig, Alex Rolfe, Olaf Christensen, Isabelle Dussault. Identification of a tumor biomarker in advanced triple-negative breast cancer that predicts response to bintrafusp alfa (M7824), a bifunctional fusion protein targeting transforming growth factor-β and programmed death ligand 1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-13.

Identification of low-dose, low-molecular-weight, drug-like inhibitors of protein-protein interactions (PPIs) is a challenging area of research. Despite the challenges, the therapeutic potential of PPI inhibition has driven significant efforts toward this goal. Adding to recent success in this area, we describe herein our efforts to optimize a novel purine carboxylic acid-derived inhibitor of the HDM2-p53 PPI into a series of low-projected dose inhibitors with overall favorable pharmacokinetic and physical properties. Ultimately, a strategy focused on leveraging known binding hot spots coupled with biostructural information to guide the design of conformationally constrained analogs and a focus on efficiency metrics led to the discovery of

Introduction/Background* The accelerated US Food and Drug Administration approval of pembrolizumab validated the efficacy of anti-PD-(L)1 therapy for patients with recurrent/metastatic cervical cancer; however, the objective response rate (ORR) with pembrolizumab was 14.3% in patients with PD-L1–expressing tumours. Human papillomavirus infection is implicated in >95% of cervical cancers and is linked to upregulation of TGF-β signalling. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β ‘trap’) fused to a human immunoglobulin G1 monoclonal antibody blocking PD-L1. We report pooled safety and efficacy in patients with pretreated, immune checkpoint inhibitor-naive, recurrent/metastatic cervical cancer treated with bintrafusp alfa in phase 1 (INTR@PID 001; NCT02517398) and phase 2 (study 012; NCT03427411) studies. Methodology Patients received bintrafusp alfa 0.3-30 mg/kg (phase 1 dose escalation) or 1200 mg every 2 weeks (phase 1 dose expansion and phase 2) until progressive disease, unacceptable toxicity, or withdrawal. Treatment past progression was allowed. Primary endpoints were safety (phase 1 dose escalation) and best overall response per RECIST 1.1 (phase 1 dose expansion and phase 2). Result(s)* As of May 15, 2020 (phase 1) and December 22, 2020 (phase 2), 39 patients had received bintrafusp alfa for a median duration of 2.8 months (range, 0.5-19.3). The median follow-up to data cutoff was 35.0 months and 24.1 months for the phase 1 and phase 2 studies, respectively. All patients had received prior anticancer therapy; 16 (41.0%) had received ≥3 regimens. Confirmed ORR was 28.2% (table 1); responses occurred irrespective of Moore criteria (phase 1), tumour histology, prior bevacizumab treatment, or radiation treatment. Median overall survival was 13.4 months. No new safety signals and no treatment-related deaths were observed; side effects were manageable. Conclusion* Bintrafusp alfa had a manageable safety profile and demonstrated clinical activity in patients with heavily pretreated, immune checkpoint inhibitor-naive recurrent/metastatic cervical cancer. © 2021 American Society of Clinical Oncology, Inc. Reused with permission. This abstract was accepted and previously presented at the 2021 ASCO Annual Meeting. All rights reserved.

Background For patients with recurrent glioblastoma (rGBM), there are few options following treatment failure with radiotherapy plus temozolomide. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 antibody blocking PD-L1. Methods In this phase I, open-label expansion cohort (NCT02517398), patients with rGBM that progressed after radiotherapy plus temozolomide received bintrafusp alfa 1200 mg Q2W until disease progression, unacceptable toxicity, or trial withdrawal. Response was assessed per RANO criteria. The primary endpoint was disease control rate (DCR); secondary endpoints included safety. Results As of August 24, 2018, 35 patients received bintrafusp alfa for a median of 1.8 (range, 0.5–20.7) months. Eight patients (22.9%) experienced disease control as assessed by an independent review committee: 2 had a partial response, 4 had stable disease, and 2 had non-complete response/non-progressive disease. Median progression-free survival (PFS) was 1.4 (95% confidence interval [CI], 1.2–1.6) months; 6- and 12-month PFS rates were 15.1% and 11.3%, respectively. Median overall survival (OS) was 5.3 (95% CI, 2.6–9.4) months; 6- and 12-month OS rates were 44.5% and 30.8%, respectively. The DCR (95% CI) was 66.7% (22.3–95.7%) for patients with IDH-mutant GBM (n = 6) and 13.8% (3.9–31.7%) for patients with IDH–wild-type GBM (n = 29). Disease control was seen regardless of PD-L1 expression. Twenty-five patients (71.4%) experienced treatment-related adverse events (grade ≥3; 17.1% [n = 6]). Conclusions The percentage of patients achieving disease control and the manageable safety profile may warrant further investigation of bintrafusp alfa in GBM.

Introduction The safety and efficacy of bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the transforming growth factor β (TGF-β) receptor II (a TGF-β “trap“) fused to a human immunoglobulin G1 antibody blocking programmed death-ligand 1 (PD-L1), was evaluated in patients with advanced NSCLC. Methods This expansion cohort of NCT02517398 , an ongoing, phase 1, open-label trial, includes 80 patients with advanced NSCLC that progressed after platinum doublet therapy or after platinum-based adjuvant or neoadjuvant treatment and those who also have not received previous immunotherapy. Patients were randomized at a one-to-one ratio to receive either bintrafusp alfa 500 mg or the recommended phase 2 dosage of 1200 mg every 2 weeks. The primary end point was the best overall response (by Response Evaluation Criteria in Solid Tumors 1.1 as adjudicated by independent review committee) and was assessed by the objective response rate (ORR). Results A total of 80 patients were randomized to receive bintrafusp alfa 500 or 1200 mg (n = 40 each). Median follow-up was 51.9 weeks (IQR, 19.6–74.0). The ORR in all patients was 21.3% (17 of 80). The ORR was 17.5% (seven of 40) and 25.0% (10 of 40) for the 500 mg dose and the 1200 mg dose (recommended phase 2 dose), respectively. At the 1200 mg dose, patients with PD-L1–positive and PD-L1–high (≥80% expression on tumor cells) had ORRs of 36.0% (10 of 27) and 85.7% (six of seven), respectively. Treatment-related adverse events occurred in 55 of the 80 patients (69%) and were graded as greater than or equal to 3 in 23 of the 80 patients (29%). Of the 80 patients, eight (10%) had a treatment-related adverse event that led to treatment discontinuation; no treatment-related deaths occurred. Conclusions Bintrafusp alfa had encouraging efficacy and manageable tolerability in patients with NSCLC previously treated with platinum.

Purpose: Patients with advanced gastric/gastroesophageal junction cancer (GC/GEJC) have limited treatment options after first-line therapy. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGFβRII receptor (a TGFβ “trap”) fused to a human IgG1 antibody against programmed death ligand 1 (PD-L1), potentially offering a new treatment approach for these patients. We report results for bintrafusp alfa in GC/GEJC. Patients and Methods: Asian patients with recurrent GC/GEJC for whom standard therapy does not exist or for whom standard therapy has failed enrolled in this expansion cohort of an ongoing phase I trial and received bintrafusp alfa 1,200 mg once every 2 weeks until disease progression, unacceptable toxicity, or withdrawal. The primary objective was to assess safety/tolerability. Results: By July 23, 2018, 31 heavily pretreated patients received bintrafusp alfa for a median of 10.1 weeks; 3 patients remained on treatment. Six patients (19%) experienced grade 3 treatment-related adverse events (AE); no grade 4 events occurred. One on-treatment death occurred (sudden death); rupture of a preexisting thoracic aortic aneurysm was the suspected cause. Ten patients (32%) had immune-related AEs. The confirmed objective response rate per independent review committee was 16%; disease control rate was 26%. Median duration of response was 8.7 months (range, 2.4–12.4+). Responses occurred irrespective of PD-L1 expression or microsatellite instability status and appeared to correlate with high tumor TGFB1 levels. Conclusions: In this first evaluation in Asian patients with heavily pretreated advanced GC/GEJC, bintrafusp alfa demonstrated a manageable safety profile and clinical activity.

Introduction/Background Globally, cervical cancer is one of the most common and lethal gynaecological cancers. Advanced/metastatic disease is typically treated with chemotherapy, often with poor objective response rates (ORRs) and durations of response (DORs) in pretreated patients; ORRs with anti-PD-1 therapies range from 12%-26%. Bintrafusp alfa* (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β ‘trap’) fused to a human IgG1 mAb blocking PD-L1. We report safety and efficacy in patients with cervical cancer treated with bintrafusp alfa in dose-escalation and expansion phases of an ongoing phase 1 trial (NCT02517398). Methodology Patients with advanced/metastatic disease received bintrafusp alfa 0.3–30 mg/kg (dose-escalation, n=10) or 1200 mg (expansion, n=15) Q2W until PD, unacceptable toxicity, or withdrawal. Treatment past PD was allowed if adverse events (AEs) were tolerable and no new treatment was indicated. Primary endpoints were safety (dose-escalation) and BOR (expansion). Results As of 9 January 2019, 25 patients were treated for a median duration of 9.6 (range, 2.0–72.0) weeks. Six patients had confirmed responses per RECIST 1.1 (ORR, 24.0%); 5/6 responses were ongoing at data cutoff (median DOR not reached; range, 2.3±24.9+ months). One additional patient had a delayed PR that was ongoing for 8.7 months at data cutoff, with 73.3% disease shrinkage after initial PD (total clinical response rate, 28.0%). Responses occurred irrespective of PD-L1 protein expression: 1 patient with PD-L1-negative disease had a response. Grade 3 treatment-related AEs (TRAEs) occurred in 6 patients (24.0%); 1 patient experienced a grade 4 TRAE (4.0%; hypokalaemia). Six patients discontinued treatment due to TRAEs. No treatment-related deaths occurred. Conclusion Bintrafusp alfa has a manageable safety profile and promising clinical activity with durable responses. Further investigation of bintrafusp alfa in cervical and other HPV-associated cancers is ongoing (NCT03427411). Updated results will be presented. Disclosure TL discloses honoraria from Foundation Medicine, Takeda, and PUMA, and receives grant/research support from Foundation Medicine, Pfizer, AstraZeneca, Hengrui, and Eureka. JS discloses inventorship on a National Institutes of Health patent related to the work. LSO and ID disclose employment with EMD Serono, Inc. CH discloses employment with Merck Healthcare KGaA. JLG discloses that National Cancer Institute has a cooperative research and development agreement with EMD Serono, Inc. and discloses inventorship on a National Institutes of Health patent related to the work. All remaining authors have nothing to disclose. Funding for this trial was provided in part by the Center for Cancer Research, National Cancer Institute, and National Institutes of Health Clinical Center. This trial was sponsored by Merck Healthcare KGaA, Darmstadt, Germany, and is part of an alliance between Merck Healthcare KGaA and GlaxoSmithKline. *Proposed INN

Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (TGF-β "trap") fused to a human IgG1-blocking PD-L1, showed a manageable safety profile and clinical activity in phase I studies in patients with heavily pretreated advanced solid tumors. The recommended phase 2 dose (RP2D) was selected based on integration of modeling, simulations, and all available data. A 1,200-mg every 2 weeks (q2w) dose was predicted to maintain serum trough concentration (Ctrough ) that inhibits all targets of bintrafusp alfa in circulation in > 95% of patients, and a 2,400-mg every 3 weeks (q3w) dose was predicted to have similar Ctrough . A trend toward an association between exposure and efficacy variables and a relatively stronger inverse association between clearance and efficacy variables were observed. Exposure was either weakly or not correlated with probability of adverse events. The selected intravenous RP2D of bintrafusp alfa is 1,200 mg q2w or 2,400 mg q3w.

5509 Background: The accelerated FDA approval of pembrolizumab validated the efficacy of anti–PD-(L)1 therapy for pts with recurrent/metastatic cervical cancer; however, the objective response rate (ORR) with pembrolizumab was 14.3% in pts with PD-L1 expressing tumors. HPV infection is implicated in > 95% of cervical cancers and is linked to upregulation of TGF-β signaling. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. We report pooled safety and efficacy in pts with immune checkpoint inhibitor–naive, recurrent/metastatic cervical cancer treated with bintrafusp alfa in phase 1 (INTR@PID 001; NCT02517398) and phase 2 (study 012; NCT03427411) studies. Methods: Pts with pretreated, immune checkpoint inhibitor–naive, recurrent/metastatic cervical cancer received bintrafusp alfa 0.3-30 mg/kg (phase 1 dose escalation) or 1200 mg Q2W (phase 1 expansion/phase 2) until progressive disease, unacceptable toxicity, or withdrawal. Treatment past progression was allowed. Primary endpoints were safety for the dose-escalation part of the phase 1 study and best overall response per RECIST 1.1 for the expansion part of phase 1 and phase 2 studies. Secondary endpoints for the expansion part of the phase 1 and 2 studies included safety. Results: As of May 15, 2020 (phase 1) and December 22, 2020 (phase 2), 39 pts had received bintrafusp alfa for a median duration of 2.8 months (range, 0.5-19.3). The median follow-up to data cutoff was 35.0 months and 24.1 months for the phase 1 and phase 2 studies, respectively. All pts had received prior anticancer therapy; 16 pts (41.0%) had received ≥3 prior anticancer regimens. There were 2 complete responses and 9 partial responses (PRs; ORR per RECIST 1.1, 28.2%). Median duration of response was 11.7 months (range, 1.4-41.2), and 5 pts (45.5%) had ongoing responses (duration 1.5-41.2 months). An additional delayed PR was observed (duration 23.7 months). Reponses occurred irrespective of tumor histology or prior bevacizumab or radiation treatment. Median overall survival (mOS) was 13.4 months (95% CI, 5.5 to not reached); 24-month OS rate was 33.2%. Any-grade treatment-related adverse events (TRAEs) occurred in 33 pts (84.6%). Grade 3 TRAEs occurred in 8 pts (20.5%; anemia, colitis, gastroparesis, upper gastrointestinal hemorrhage, keratoacanthoma, cystitis noninfective, hematuria, pneumonitis, rash macular [n = 1 each]); 1 patient (2.6%) had a grade 4 TRAE (asymptomatic hypokalemia related to the above grade 3 gastroparesis). No treatment-related deaths occurred. Conclusions: Bintrafusp alfa had a manageable safety profile and demonstrated clinical activity in pts with heavily pretreated, immune checkpoint inhibitor–naive recurrent/metastatic cervical cancer. Clinical trial information: NCT02517398 , NCT03427411.

Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp–mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998–2008. ©2016 AACR.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Background: Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor, to function as a TGF-β “trap”, fused to a human IgG1 antibody blocking PD-L1, which has shown early evidence of clinical activity in phase 1 studies in patients with advanced solid tumors. We recently reported high expression of HMGA2 in patients with triple negative breast cancer (TNBC) who experienced disease control (response or stable disease) with bintrafusp alfa treatment compared with those who had progressive disease, suggesting that HMGA2 may predict response to bintrafusp alfa in this indication. HMGA2 is a transcriptional regulator whose expression is upregulated by TGF-β signaling and is known to be an important factor in mediating TGF-β–induced epithelial–mesenchymal transition (EMT). The purpose of this study was to evaluate the association between HMGA2 expression and TGF-β signaling in TNBC and the effect of bintrafusp alfa on HMGA2/TGF-β signaling. Methods: The syngeneic murine 4T1 tumor model was utilized for this study because it closely mimics human stage IV TNBC, has high baseline levels of HMGA2 expression, and bintrafusp alfa directly modulates canonical TGF-β signaling and induces antitumor activity in this model. Results: Tumors from mice treated with isotype control, anti–PD-L1, anti–PD-L1(mut)/TGF-β trap (trap control), or bintrafusp alfa were analyzed by RNAseq and both bintrafusp alfa or trap control treatment decreased HMGA2 expression relative to anti–PD-L1 or isotype control treatment. In isotype control-treated 4T1 tumors Spearman's rho correlation coefficient showed that 29% (26/89) of the TGF-β signaling-related genes significantly correlated with HMGA2 expression, indicating that there is a strong association between HMGA2 and TGF-β signaling. In bintrafusp alfa-treated tumors, the association of HMGA2 and TGF-β signaling was even stronger, with 55% (49/89) of TGF-β signaling-related genes significantly associated with HMGA2, suggesting that there is a relationship between HMGA2, TGF-β signaling, and bintrafusp alfa pharmacodynamic effects in this TNBC model. Analysis of individual TGF-β signaling-related genes from this signature showed significant correlation between HMGA2 expression and the expression of TGF-β receptors, ligands, collagen, and EMT-related genes. Furthermore, both bintrafusp alfa and trap control treatment significantly reduced expression of these HMGA2-correlated TGF-β signaling-related genes relative to anti–PD-L1 or isotype control treatment in the 4T1 model, suggesting that HMGA2 is associated with TGF-β-related signaling more strongly in the presence of TGF-β sequestration. Conclusions: Taken together, these observations warrant further analysis of the potential link between HMGA2 and bintrafusp alfa antitumor efficacy. Citation Format: Tsz-Lun Yeung, George Locke, Adam Lazorchak, Guozhong Qin, Huakui Yu, Jin Qi, Bo Marelli, Molly Jenkins, Alex Rolfe, Laureen S. Ojalvo, Isabelle Dussault, Yan Lan. Association between TGF-β signaling and HMGA2, a potential biomarker for bintrafusp alfa in triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1573.

Background: Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In expansion cohorts of global, phase 1, open-label trials (NCT02517398 and NCT02699515), extensive analysis was conducted to identify potential tumor biomarkers correlated with bintrafusp alfa efficacy. Methods: Immunohistochemistry was performed on archival or fresh formalin-fixed and paraffin-embedded tumor samples using anti-PD-L1 antibody clone 73-10. Based on H&E and PD-L1 staining with negative controls, tumors were scored as having an immune desert ( Results: The frequency of immune phenotypes varied by tumor type. Triple negative breast cancer (TNBC) and biliary tract cancer (BTC) had no/few inflamed tumors (0/30 and 2/28, respectively) while gastric cancer (GC) and esophageal squamous and adenocarcinoma cancers (ESCC and ESAD) had few immune desert tumors (GC, 1/30; ESCC, 1/30; ESAD, 1/27). Response to bintrafusp alfa varied by immune phenotype. The majority of responders to bintrafusp alfa in GC and squamous cell carcinoma of the head and neck (SCCHN) had inflamed phenotypes (4/5 and 3/4, respectively) while non-small cell lung cancer (NSCLC) responders had similar numbers of inflamed (7/17) and immune excluded (9/17) phenotypes. Most responders to bintrafusp alfa in ESCC, ESAD, TNBC, and BTC had immune excluded tumors (3/3, 5/6, 2/3, 5/6, respectively). Responses rarely occurred in immune desert tumors (n=1 each in NSCLC, BTC, and TNBC). Evaluation of genes/gene signatures found inflamed tumors had a significantly higher level of immune genes/gene signatures compared to immune excluded or immune desert tumors. In TNBC and ESCC there were significantly higher levels of some genes related to TGF-β biology in immune excluded tumors compared to the other two phenotypes. This potential link to TGF-β biology was less clear in other tumor types. Immune excluded and inflamed tumors could not be distinguished by molecular methods across all indications. Conclusion: The data suggest that both pathology evaluation of immune phenotypes and molecular methods are useful to understand response to bintrafusp alfa. Moreover, several complementary biomarkers are likely needed to more precisely predict response to bintrafusp alfa, a drug designed to have a bifunctional mechanism of action, across cancer indications. Citation Format: George Locke, Christian Ihling, Christoph Helwig, Laureen S. Ojalvo, Olaf Christensen, Isabelle Dussault. Evaluation of immune phenotypes and gene expression profiles in tumors from patients treated with bintrafusp alfa [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3290.

9558 Background: Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. Interim analysis of a global phase 1 study (NCT02517398) found an objective response rate (ORR) of 27.5% and a manageable safety profile in patients with NSCLC who received bintrafusp alfa 1200 mg in the 2L setting; median overall survival (OS) was not reached. Here we present the longest efficacy and safety follow-up in a cohort receiving bintrafusp alfa. Methods: Patients with advanced NSCLC unselected for PD-L1 expression level who progressed after first-line standard treatment (no prior immunotherapy) were randomized to receive bintrafusp alfa 500 or 1200 mg (n = 40 each) Q2W until disease progression, unacceptable toxicity or trial withdrawal. The primary objective was best overall response (BOR) per RECIST 1.1; secondary and exploratory objectives include safety and OS, respectively. Results: As of October 15, 2019, a total of 40 patients received bintrafusp alfa at the recommended phase 2 dose of 1200 mg Q2W for a median of 17 (range, 2-136) weeks, with a median follow-up of 128 weeks; 18 patients were still alive, 3 patients had an ongoing response, and 1 patient remained on treatment. Results for the 1200 mg dose cohort showed an ORR of 27.5%, and a median duration of response of 18 months. The 18- and 24-month progression-free survival and OS rates were 18.4% and 11.0%, and 49.7% and 39.7%, respectively. Duration of response rates at 18 and 24 months were 42.4% and 21.2%, respectively. Median OS in patients with positive (≥1%) PD-L1 expression was 21.7 months; 6 of 7 patients with high (≥80% with Ab clone 73-10, which is equivalent to ≥50% with 22C3) PD-L1 expression were still alive. The overall safety profile has remained consistent since the interim analysis, with no new safety signals or deaths and 1 additional treatment-related discontinuation (blood alkaline phosphatase increased). Conclusions: After two years of follow-up, bintrafusp alfa continues to show manageable safety with durable responses and encouraging long-term survival, especially in patients with high PD-L1 expression. A study evaluating bintrafusp alfa vs pembrolizumab as first-line treatment for NSCLC is ongoing in patients with high PD-L1 expression (NCT03631706). Clinical trial information: NCT02517398 .

Introduction: Hepatocellular carcinoma (HCC) typically develops in cirrhotic livers, with increased programed death ligand 1 (PD-L1) and transforming growth factor beta (TGF-β) activity implicated in immunosuppression. Methods: In an observational study of HCC liver samples, we determined the incidence of PD-L1 and immune cell (IC) infiltrates, and signs of TGF-β activity. HCCs were characterized by the incidence and distribution of PD-L1+ cells, and CD8+, CD68+, and FoxP3+ infiltrating ICs in HCC and surrounding liver. Gene expression signatures (GESs) associated with TGF-β activity and ICs were evaluated by RNAseq. Results: In non-neoplastic cirrhotic and non-cirrhotic liver, PD-L1 occurred on sinusoidal lining cells (mostly Kupffer cells), endothelial cells and ICs. In HCC, PD-L1+ tumor cells were rare. Most PD-L1+ cells were identified as ICs. CD8+, CD68+, and FoxP3+ ICs were associated with HCC, particularly in the invasive margin. CD8+ cell incidence correlated with PD-L1+ cells, consistent with PD-L1 being upregulated in response to pre-existing cytotoxic T-lymphocyte activity. TGFB1 mRNA levels and TGF-β activation GES correlated with the strength of the tumor-associated macrophage GES. Conclusion: Inhibition of PD-L1+ ICs and TGF-β activity and their respective immunomodulatory pathways may contribute to antitumor effects in HCC.

Contains fulltext : 181795.pdf (Publisher’s version ) (Closed access) The receptor tyrosine kinase (RTK) MET represents a promising tumor target in a subset of glioblastomas. Most RTK inhibitors available in the clinic today, including those inhibiting MET, affect multiple targets simultaneously. Previously, it was demonstrated that treatment with cabozantinib (MET/VEGFR2/RET inhibitor) prolonged survival of mice carrying orthotopic patient-derived xenografts (PDX) of the MET-addicted glioblastoma model E98, yet did not prevent development of recurrent and cabozantinib-resistant tumors. To exclude VEGFR2 inhibition-inflicted blood-brain barrier normalization and diminished tumor distribution of the drug, we have now investigated the effects of the novel MET-selective inhibitor Compound A in the orthotopic E98 xenograft model. In vitro, Compound A proved a highly potent inhibitor of proliferation of MET-addicted cell lines. In line with its target selectivity, Compound A did not restore the leaky blood-brain barrier and was more effective than cabozantinib in inhibiting MET phosphorylation in vivo Compound A treatment significantly prolonged survival of mice carrying E98 tumor xenografts, but did not prevent eventual progression. Contrasting in vitro results, the Compound A-treated xenografts displayed high levels of AKT phosphorylation despite the absence of phosphorylated MET. Profiling by RNA sequencing showed that in vivo transcriptomes differed significantly from those in control xenografts.Implications: Collectively, these findings demonstrate the plasticity of paracrine growth factor receptor signaling in vivo and urge for prudency with in vitro drug-testing strategies to validate monotherapies. Mol Cancer Res; 15(11); 1587-97. (c)2017 AACR.