Tracon Pharmaceuticals (United States)

Liver sinusoidal endothelial inflammation/dysfunction and fibrosis are a crucial part of Metabolic Dysfunction Associated Steatohepatitis (MASH) development. TRC105 and M1043 are anti-endoglin (ENG) monoclonal antibodies that bind ENG. In this study, we hypothesized that treatment with anti-ENG antibodies would prevent the progression of LSECs inflammation and fibrosis in vivo and in vitro. MASH was induced in male C57BL/6 mice fed a choline-deficient L-amino acid-defined high-fat diet (CDAA-HFD) for 4 or 8weeks. In the rescue study, mice were divided into three groups: a control group (chow diet), a MASH group (CDAA-HFD+IgG), and a rescue group (CDAA-HFD+M1043). Later, two groups received rat IgG1 (10mg/kg) and M1043 (10mg/kg). In in vitro experiments, inflammation was induced in human LSECs by ox-LDL (50μg/mL) and treated with TRC105 (300μg/mL). Liver sinusoidal endothelial inflammation/dysfunction in MASH animals was characterized by endothelial overexpression of ENG, VCAM-1, and ICAM-1 and reduced VE-cadherin and p-eNOS/eNOS expression. M1043 treatment prevented the overexpression of ENG, VCAM-1, and ICAM-1, the progression of liver fibrosis, and the increase of liver-to-body weight ratio. In vitro experiments with TRC105 confirmed the prevention of LSECs inflammation development by reduced ENG and VCAM-1 expression, as well as decreased THP-1 monocytic cell adhesion in ox-LDL activated LSECs. In conclusion, we demonstrate that anti-ENG antibody treatment can prevent LSECs inflammation and fibrosis progression in a MASH animal model and LSECs inflammation in vitro. Thus, we propose directly targeted ENG may represent a promising pharmacological approach for addressing LSECs inflammation and liver fibrosis.

ABSTRACTPurposeMalignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive softtissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGF-β coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs.Experimental DesignENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathways and in vivo MPNST growth and metastasis were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models.ResultsENG expression was found to be upregulated in both human MPNST tumor tissues and plasma circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST tumor growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Importantly, combination of anti-ENG therapy with MEK inhibition reduced more effectively tumor cell growth and angiogenesis.ConclusionsOur data unveil a tumor-promoting function of ENG in MPNSTs and support the combined use of anti-ENG antibodies and MEK inhibitors as a novel potential combination to control MPNSTs growth and metastasis.Statement of translational relevanceMalignant peripheral nerve sheath tumors (MPNSTs) present a poor clinical outcome due to tumor aggressiveness and the absence of effective treatments that underline the need for identifying novel therapeutic approaches. Here, we show that ENG is upregulated in human MPNSTs and we uncover an important role for this coreceptor in MPNST. ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST tumor growth and metastasis in xenograft models, supporting a novel use of anti-ENG therapies for MPNST treatment. Currently, MEK inhibitors are used in the clinic to shrink peripheral nerve sheath tumors (e.g. plexiform neurofibromas). Importantly, we demonstrate that combination of anti ENG antibodies with MEK inhibitors efficiently blocked MPNST growth and metastasis. Our findings provide a rationale for combining anti-ENG and MEK inhibitors as a new strategy for MPNST management.

Cancer-associated fibroblasts (CAFs) are the major component of the tumor microenvironment in colorectal cancer (CRC) and play an important role in tumor progression and metastasis, partly through the transforming growth factor-β (TGF-β) signaling pathway. Recently, we showed that a subset of CAFs characterized by the expression of the TGF-β family co-receptor endoglin, selectively localized at the invasive borders of CRC tissues. Moreover, CAF-specific endoglin expression increased with higher tumor stage and predicted metastasis-free survival of stage-II CRC patients (n= 140, p<0.05). Targeting endoglin on CAFs using TRC105 (carotuximab), an endoglin neutralizing antibody currently in clinical development, inhibits endoglin dependent signaling and metastatic colonization when CRC cells and CAFs are co-injected into mice. We further investigated these anti-tumor effects in four different models of CRC (chemically-induced model of early CRC, subcutaneous MC38 and CT26 syngeneic mouse CRC models, and orthotopic MC38 model). TRC105 treatment inhibited tumor initiation (early stage CRC), growth, and progression. Therapeutic effects of TRC105 therapy were dependent on antibody dependent cytotoxicity (ADCC) and CD8+ T-cells, as no therapeutic benefit from TRC105 was observed in Fc-y I-IV receptor knockout mice or CD8+ cell depleted mice. Since therapeutic TRC105 activity was immune cell dependent, we combined TRC105 with anti-programmed cell death (PD-1 immunotherapy). We observed synergistic inhibition of tumor initiation and growth in all four tumor models, leading to delayed tumor outgrowth, increased survival and cure rates up to 60% in the combination treated mice bearing palpable tumors. Combined treatment also induced memory responses, as shown by resistance to repeated tumor challenge. Anti-tumor responses were accompanied by enhanced immune cell activation including an increased number of tumor-specific T-cells, NK cells, decreased number of tumor-infiltrating neutrophils and increased granzyme B production. CD8+ cytotoxic T-cells were instrumental in these synergistic therapeutic responses, as their depletion abolished the effects. Taken together, our data indicate significant therapeutic effects of targeting endoglin by affecting signaling and multiple immune related components in the tumor microenvironment. These results are currently being translated in a clinical trial combining TRC105 with nivolumab in patients with non-small cell lung cancer (NCT03181308).Citation Format: Mark JA Schoonderwoerd, Madelon Paauwe, Ricardo Angela, Maaike Koops, Tom Harryvan, Cornelis Sier, Marieke Fransen, Charles Theuer, Lukas JAC Hawinkels. Synergistic inhibition of cancer invasion and metastasis by combined anti-PD1-TRC105-mediated Endoglin targeting on cancer-associated fibroblasts and endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 291.

Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis. CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer-derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model. CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9-induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver. Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations.See related commentary by Becker and LeBleu, p. 6110.

TRC105 is an anti-endoglin antibody currently being tested in combination with VEGF inhibitors. In the phase Ib trial, 38 patients were treated with both TRC105 and bevacizumab (BEV), and improved clinical outcomes were observed, despite the fact that 30 patients (79%) were refractory to prior anti-VEGF therapy. Plasma samples were tested for angiogenic and inflammatory biomarkers at baseline and on-treatment. To provide broader context of this combination biomarker study, direct cross-study comparisons were made to biomarker studies previously conducted in patients treated with either BEV or TRC105 monotherapy. Upon treatment with BEV and TRC105, pharmacodynamic changes in response to both BEV (PlGF increase) and TRC105 (soluble endoglin increase) were noted. In addition, distinct patterns of change were identified (similar, opposing, neutralizing). Similar patterns were observed when the combination elicited similar effects to those observed with monotherapy treatment (i.e., decreases of Ang-2, increases of IL6 and VCAM-1). Opposing patterns were observed when the combination led to opposing effects compared with monotherapy treatment (i.e., TGFβ1, PDGF-AA and PDGF-BB, PAI-1). Lastly, neutralizing patterns were observed when one drug led to increase, whereas the other drug led to decrease, and the combination elicited no overall effect on the marker (i.e., VEGF-A, VEGF-D, and IGFBP-3). Patients achieving partial responses or stable disease from the combination exhibited significantly lower expression of E-Cadherin, HGF, ICAM-1, and TSP-2 at baseline. Taken together, the novel biomarker modulations identified may deepen our understanding of the underlying biology in patients treated with BEV and TRC105 compared with either drug alone. Mol Cancer Ther; 17(10); 2248-56. ©2018 AACR.

Angiogenesis is widely recognized as one of the hallmarks of cancer. Therefore, imaging and therapeutic agents targeted to angiogenic vessels may be widely applicable in many types of cancer. To this end, the theranostic isotope pair, 86Y and 90Y, were used to create a pair of agents for targeted imaging and therapy of neovasculature in murine breast cancer models using a chimeric anti-CD105 antibody, TRC105. Serial positron emission tomography imaging with 86Y-DTPA-TRC105 demonstrated high uptake in 4T1 tumors, peaking at 9.6 ± 0.3%ID/g, verified through ex vivo studies. Additionally, promising results were obtained in therapeutic studies with 90Y-DTPA-TRC105, wherein significantly ( p < 0.05) decreased tumor volumes were observed for the targeted treatment group over all control groups near the end of the study. Dosimetric extrapolation and tissue histological analysis corroborated trends found in vivo. Overall, this study demonstrated the potential of the pair 86/90Y for theranostics, enabling personalized treatments for cancer.

TPS403 Background: Resistance to AR-targeted therapy is a challenge in the treatment of mCRPC. Single amino acid mutations of the AR ligand binding domain may mediate resistance to second generation AR inhibitors, including enzalutamide (Rathkopf, Annals of Oncology 2017). Development of potent antagonists of wild-type (WT) AR as well as mutated AR is a priority. TRC253 is an orally available, high-affinity, small molecule antagonist of AR with inhibitory activity against WT AR as well as mutated variants. TRC253 blocks AR nuclear translocation and AR binding to DNA and antagonizes transcription. TRC253 does not have agonist activity toward WT or mutated ARs. Methods: This phase 1/2a study of TRC253 in patients with mCRPC will be conducted in two parts: dose-escalation (part 1) and dose-expansion (part 2). Objectives include assessment of safety, selection of a phase 2 dose, and to evaluate PSA response at week 12. Secondary objectives include the evaluation of the extent of receptor occupancy (FDHT PET) and anti-tumor effects of TRC253. Toxicity and efficacy assessments will be determined using NCI-CTCAE and PCWG3 criteria. Dose escalation will begin with single-patient cohorts. Cohorts will be expanded to 3 patients when pre-defined grades of drug-related toxicity occur. TRC253 doses in part 1 are 40 mg, 80 mg, 160 mg, 240 mg, 320 mg, and 400 mg. The DLT evaluation period will be the first 28 days of continuous daily dosing. Six patients have been enrolled to Part 1 to date. Part 2 will consist of two cohorts of up to 30 patients each. Cohort 1 (AR F876L mutation positive) and cohort 2 (AR F876L mutation negative) will receive TRC253 at the RP2D. Circulating tumor DNA will be analyzed in plasma samples to test for AR mutations. In Part 1 patients must have received ≥ 2 prior therapies. Part 2 patients must have demonstrated acquired resistance to enzalutamide or apalutamide. Descriptive statistics will be used to summarize patient characteristics, safety, efficacy, PK, and immunologic biomarkers. Clinical trial information: NCT02987829.

Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab' antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide (64Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF+/+) or PANC-1 (CD105/TF-/-) tumor xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of 64Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4%ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0%ID/g; n = 4; p < 0.01) and ALT-836 (7.6 ± 3.7%ID/g; n = 4; p < 0.01) at 24 h postinjection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by 4-fold with TF blocking and 2-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1%ID/g; n = 4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated that the dual targeting with 64Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like 64Cu-NOTA-heterodimer-ZW800, may increase the overall tumor accumulation in comparison to single-targeted homodimers, leading to improved imaging of cancer and other related diseases.