TGF-β Trap Research Articles

The Variation in the Traits Ameliorated by Inhibitors of JAK1/2, TGF-β, P-Selectin, and CXCR1/CXCR2 in the Gata1low Model Suggests That Myelofibrosis Should Be Treated by These Drugs in Combination.

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-β (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-β) signaling and TGF-β trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-β content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-β or CXCL1).

Abstract CT264: Improved clinical outcomes in patients received treatment beyond tumor progression with AdAPT-001 +/- a checkpoint inhibitor

Abstract Background AdAPT-001 is an oncolytic adenovirus with a TGF-β trap. Pseudoprogression (PsP) or transient tumor enlargement from intense T cell infiltration was commonly observed with AdAPT-001 in immune competent mouse models. We evaluated progression-free survival (PFS) and overall survival (OS) in 36 treatment-resistant patients (pts) that received AdAPT +/- an ICI on a Phase 2 clinical trial. Methods RECIST1.1 assessment was performed every 8 weeks. PsP was defined as progressive disease (PD) on RECIST 1.1 with subsequent tumor regression during TBP. The decision to TBP was based on investigator-assessed clinical benefit and the potential for late responses with minimal adverse events. Log rank tests were applied to compare PFS between pts with and without PsP and to compare OS between pts with and without TBP. Results Median (m) PFS was longer in pts with PsP and TBP (3.7, 95% CI: 1.6 - ) than without it (2.0, 95% CI: 1.8 - 3.7, log-rank test p = 0.97). mOS months was significantly longer in pts with TBP (median not reached) vs. no TBP (7.8, 95% CI: 6.1 - log-rank test p = 0.053). Conclusion A high frequency of PsP occurred on this Phase 2 study likely from infiltration of treatment-activated immune cells. AdAPT-001 mediated PsP and TBP may serve as markers of prolonged clinical benefit. Further validation in larger numbers of future patients is warranted. Citation Format: Anthony P. Conley, Tony Reid, Chris Larson, Bryan Oronsky, Scott Caroen, Meaghan Stirn, Erica Burbano, Nacer Abrouk, Christina L. Roland, Jeannie Williams, Lucy B. Kennedy. Improved clinical outcomes in patients received treatment beyond tumor progression with AdAPT-001 +/- a checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT264.

A phase 1, first-in-human, open-label, dose escalation and dose expansion study of TST005 in patients with locally advanced or metastatic solid tumors.

2531 Background: TST005 is a novel bi-functional fusion protein combining a high affinity PD-L1 mAb and a differentiated TGF-β trap with improved stability. In pre-clinical study, TST005 showed no antibody-dependent dependent cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) activity. Methods: This is a Phase 1, dose escalation study with classic 3+3 design to evaluate the safety, tolerability, PK and pharmacodynamic and preliminary anti-tumor activity of TST005 and determine the maximum tolerated dose and/or the Recommended Phase 2 Dose (RP2D). Eligible solid tumor patients who have failed prior standard therapy(ies) and have received no more than one prior immune checkpoint inhibitor (ICI) treatment will be enrolled to receive TST005 intravenous (IV) infusion every 3 weeks until disease progression assessed by the Investigator per RECIST v1.1 and/or immune RECIST, or unacceptable toxicity. Results: The study is ongoing at 4 clinical centers in US and China. As of 31 Jan 2023, 18 eligible subjects have been enrolled into different TST005 dose levels: 2 at dose of 1mg/kg, 4 at 3 mg/kg, 4 at 10 mg/kg, 3 at 20 mg/kg, and 5 at the last dose level of 30 mg/kg. The enrollment is still ongoing. No DLT was observed so far. The most common treatment-emergent adverse events (TEAEs) ( > 10%) included: fatigue (38.9%), rash (22.2%), anemia (16.7%), myalgia (16.7%), and arthralgia, dehydration diarrhea, dry skin, hematuria, hypokalemia, pruritus, lymphocyte count decreased were 11.1%. 7 patients (38.9%) experienced ≥Grade 3 (G3) TEAEs according to CTCAE V5.0, 1 of whom were considered as related to TST005: it was an infusion related reaction (G3), and caused discontinuation of treatment. Two subjects experienced IRRs (G2 and G3 respectively), both at the dose of 3mg/kg cohort. One subject of 30mg/kg dose cohort presented a squamous cell carcinoma of skin, which is known as a classic side effect of TGF-β directed therapies. The subject was discontinued from study and to receive resection. No treatment-related deaths occurred. TST005 demonstrated a nonlinear PK profile between 1 to 20 mg/kg with calculated t1/2 of 4-9 days. ADA was observed in most ADA evaluable subjects, PK at doses ≥ 10mg/kg were not impacted by ADA. At 20mg/kg, the receptor occupancy increased to > 90% immediately after infusion and remain at this level up to C2D1. Start from 1mg/kg, serum TGF-β1 was depleted immediately after dosing, sustained to next dose of TST005. 11 subjects had at least one post treatment tumor assessment per RECIST 1.1. 7 subjects (63.6%) had best overall response of SD. No PR or CR had been observed. Conclusions: TST005 monotherapy demonstrated a good safety and tolerability, nonlinear PK characteristics and significant target engagement in patients with advanced solid tumors. Continue investigation of TST005 as monotherapy or in combination with other therapies are warranted. Clinical trial information: NCT04958434 .

Bintrafusp alfa, an anti-PD-L1:TGF-β trap fusion protein, in patients with ctDNA-positive, liver-limited metastatic colorectal cancer.

Identification of circulating tumor DNA (ctDNA) following curative intent therapies is a surrogate for microscopic residual disease for patients with metastatic colorectal cancer (mCRC). Preclinically, in micrometastatic microsatellite stable (MSS) CRC, increased TGF-β signaling results in exclusion of anti-tumor cytotoxic T cells from the tumor microenvironment. Bintrafusp alfa (BA) is a bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor ("TGF-β trap") and anti-PD-L1 antibody. Patients with liver-limited, MSS mCRC and with detected ctDNA after complete resection of all known tumors and standard-of-care therapy were treated with 1200 mg of BA intravenously every 14 days for six doses. The primary endpoint was ctDNA clearance. Radiographic characteristics at recurrence were compared using independent t-tests to historical data from a similar cohort of patients with liver-limited mCRC who underwent observation. Only 4 of 15 planned patients received BA before the study was stopped early for loss of equipoise. There was no grade ≥3 AE. None of the patients cleared ctDNA. All patients developed radiographic recurrence by the first planned restaging. Although not detectable at prior to treatment, TGFβ3 was found in circulation in all patients at cycle 2 day 1. Compared to a historical cohort, patients administered BA developed more metastases (15 versus 2, p=0.005) and greater tumor volumes (9 cm vs 2 cm, p=0.05). Treatment with BA in patients with ctDNA-detected, liver-limited mCRC did not clear ctDNA and was associated with large-volume recurrence, highlighting the potential context-specific complexity of dual TGF-β and PD-L1 inhibition.

Colocalized targeting of TGF-β and PD-L1 by bintrafusp alfa elicits distinct antitumor responses

BackgroundBintrafusp alfa (BA) is a bifunctional fusion protein designed for colocalized, simultaneous inhibition of two immunosuppressive pathways, transforming growth factor-β (TGF-β) and programmed death-ligand 1 (PD-L1), within the tumor microenvironment...

Abstract 6013: TST005, a bifunctional fusion protein of PD-L1/TGF-βRII, demonstrates potent anti-tumor activities with good safety profiles

Abstract Antibodies targeting PD1/PD-L1 are emerging as effective cancer immunotherapies, however, not all patients with PD-L1 expression respond to the treatment. A growing body of data demonstrates the pivotal roles of the master regulators from the tumor microenvironment (TME), such as transforming growth factor β (TGF-β), in the development of resistance to Immune checkpoint inhibitors. Therefore, co-inhibition of TGF-β signaling is expected to enhance the antitumor responses of PD1/PD-L1-based immunotherapies. TST005 is a bifunctional fusion protein composed of the truncated extracellular domain of the TGF-βRII receptor (a TGF-β trap) fused to a humanized anti-PD-L1 IgG1 antibody with ablated Fc immune effector function. Previously we showed the potent antitumor activity of TST005 in MC38/hPD-L1 and EMT6/hPD-L1 xenograft models. Here we report TST005 anti-tumor activities in MC38 colorectal cancer and EMT-6 breast cancer models compared to M7824 (Merck’s PD-L1/TGF-βRII) analog and its safety profiles following single or repeated doses in rats and non-human primates (NHP). TGF-β1depletion from the plasma of MC38 tumor-bearing mice was observed even at the lowest dose (1mg/kg). At higher dose (10mg/kg), TGF-β1 depletion persisted longer. The same trend was also found in MC38 tumor tissue. In EMT-6 tumor model upon TST005 treatment (30mg/kg), TST005 showed better tumor inhibition compared to M7824 analog at high dose (30mg/kg), which correlates well with the extent of TGF-β1depletion. For TST005, TGF-β1depletion was persistent throughout all time points tested (up to Day 14) in plasma and tumor tissue compared to M7824 analog at the same dose/dosing frequency. TST005 treatment (3mg/kg,10mg/kg) significantly lowered phosphorylated SMAD2 (pSMAD2) in MC38 tumors. TST005 was well tolerated in both rats and monkeys, no significant safety issues were observed following single or repeated doses in general toxicity studies and safety pharmacology assessment, the exposure of TST005 in both species was comparable between two genders and increased in an approximately dose-proportional manner. No cytokine release was observed in monkeys. Based on the exposure of TST005 in monkeys and predicted exposure at First in Human (FIH) dose, the safety margin of TST005 will be higher than 200 and 500 folds calculated on Cmax and AUC respectively. In conclusion, we have demonstrated the antitumor activity of TST005 in PD/PD-L1 sensitive and resistant tumor models as well as the safety profile in NHP, TST005 has been granted for phase 1 clinical trials in USA (NCT04958434). Citation Format: Huanhuan Guo, Xinlai Yao, Chunming Wang, Linlin Mao, Hongjun Li, Shuang Lu, Di Sun, Chengwei Jiang, Shenjie Zhang, Lisa Zheng, Fan Zhang, Fei Teng, Steven Yu, Yi Gu, Xueming Qian. TST005, a bifunctional fusion protein of PD-L1/TGF-βRII, demonstrates potent anti-tumor activities with good safety profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6013.

Dual drug delivery collagen vehicles for modulation of skin fibrosis in vitro

Single molecule drug delivery systems have failed to yield functional therapeutic outcomes, triggering investigations into multi-molecular drug delivery vehicles. In the context of skin fibrosis, although multi-drug systems have been assessed, no system has assessed molecular combinations that directly and specifically reduce cell proliferation, collagen synthesis and transforming growth factor β1 (TGFβ1) expression. Herein, a core–shell collagen type I hydrogel system was developed for the dual delivery of a TGFβ trap, a soluble recombinant protein that inhibits TGFβ signalling, and Trichostatin A (TSA), a small molecule inhibitor of histone deacetylases. The antifibrotic potential of the dual delivery system was assessed in an in vitro skin fibrosis model induced by macromolecular crowding (MMC) and TGFβ1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography analyses revealed that ∼50% of the TGFβ trap and ∼30% of the TSA were released from the core and shell compartments, respectively, of the hydrogel system after 10 d (longest time point assessed) in culture. As a direct consequence of this slow release, the core (TGFβ trap)/shell (TSA) hydrogel system induced significantly (p < 0.05) lower than the control group (MMC and TGFβ1) collagen type I deposition (assessed via SDS-PAGE and immunocytochemistry), α smooth muscle actin (αSMA) expression (assessed via immunocytochemistry) and cellular proliferation (assessed via DNA quantification) and viability (assessed via calcein AM and ethidium homodimer-I staining) after 10 d in culture. On the other hand, direct TSA-TGFβ supplementation induced the lowest (p < 0.05) collagen type I deposition, αSMA expression and cellular proliferation and viability after 10 d in culture. Our results illustrate the potential of core–shell collagen hydrogel systems for sustained delivery of antifibrotic molecules.

Simultaneous targeting of TGF-β/PD-L1 synergizes with radiotherapy by reprogramming the tumor microenvironment to overcome immune evasion.

Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor β (TGF-β). We report that a bifunctional fusion protein that simultaneously inhibits TGF-β and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA+ RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8+ Tcells. Intriguingly, targeting of the TGF-β trap to PD-L1+ endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation.

Abstract 917: The preclinical characterization of TST005, a bi-functional anti-PD-L1 and TGF-β trap fusion protein

Abstract Treatment with immune checkpoint inhibitors, including anti-PD(L)-1 antibodies, has demonstrated durable responses in patients with PD-L1 expressing tumors. However, not all patients with PD-L1 expression respond to the treatment due to de novo or acquired resistance to checkpoint inhibitors. The evidence from a number of studies showed that transforming growth factor β (TGF-β) signaling in the tumor microenvironment is associated with a poor response or resistance, and inhibition of TGF-β signaling can induce tumor T cell infiltration/activation and potentiate tumor response to immune checkpoint therapy. TST005 is a bifunctional fusion protein composed of the truncated extracellular domain of the TGF-βRII receptor (a TGF-β trap) fused to a humanized anti-PD-L1 IgG1 antibody (AM4B6 mAb) with ablated Fc immune effector function. TST005 bound to human/cyno PD-L1 with high affinity and blocked the PD-1/PD-L1 interaction potently in a cell-based reporter assay. Similarly, TST005 also bound to TGF-β1, 2 and 3 isoforms with high affinity and inhibited the activation of TGF-β receptor mediated signaling. In a superantigen stimulation assay with human peripheral blood mononuclear cells (PBMCs), TST005 enhanced T cell activation significantly as measured by interferon-γ (IFN-γ) production, as compared to TGF-β trap or AM4B6 mAb alone. In contrast, TST005 did not exert any cytokine release effect on human naïve PBMCs in vitro. TST005 showed a dose-proportional linear PK profile with single IV infusion in mice, and a negative correlation between plasma TST005 concentration and its pharmacodynamic marker TGF-β1 level was established. Furthermore, in the MC38/hPD-L1 xenograft mouse model, compared with TGF-β trap or AM4B6 mAb, TST005 increased the infiltration of activated CD8+ T cells into the tumor, and it resulted in tumor regression starting from 3mg/kg. Of particular interest, in the EMT6/hPD-L1 xenograft model which responds only moderately to PD-L1 inhibitors, TST005 induced significant tumor growth inhibition and regression starting from 10mg/kg. In conclusion, we have demonstrated that TST005 has enhanced immunomodulatory properties and can induce potent antitumor activity in preclinical tumor models that are not sensitive to PD-1/PD-L1 monotherapy. These results provide the rationale for further clinical evaluation of TST005 in patients with advanced solid tumors and less optimal response to first generation PD(L)-1 based immunotherapy. Citation Format: Hongjun Li, Chunming Wang, Huanhuan Guo, Xinlai Yao, Weiwei Sun, Di Sun, Yupeng Zhu, Xiangyu Xu, Zhenzhi Yan, Chengwei Jiang, Shengjie Zhang, Xiwen Wu, Yingdi Luo, Yadong Dai, Lisa Zheng, Fan Zhang, Fei Teng, Xueming Qian, Yi Gu. The preclinical characterization of TST005, a bi-functional anti-PD-L1 and TGF-β trap fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 917.

Secretion of bispecific protein of anti-PD-1 fused with TGF-β trap enhances antitumor efficacy of CAR-T cell therapy

Despite the remarkable success of chimeric antigen receptor-modified T (CAR-T) cell therapy for blood malignancies, the clinical efficacy of this novel therapy in solid tumor treatment is largely limited by the immunosuppressive tumor microenvironment (TME). For instance, immune checkpoints (e.g., programmed cell death protein 1 [PD-1]/programmed death ligand 1 [PD-L1]) in TME play an important role in inhibiting T cell proliferation and functions. Transforming growth factor β (TGF)-β secreted by cancer cells in TME induces regulatory T cells (Tregs) and inhibits cytotoxic T cells. To overcome the inhibitory effect of immune checkpoints, we have previously engineered CAR-T cells to secrete anti-PD-1 to block the PD-1/PD-L1 pathway activity, a step demonstrating superior antitumor efficacy compared with conventional CAR-T cells. In this study, we engineered CAR-T cells that secrete bispecific trap protein co-targeting PD-1 and TGF-β, with the aim of further improving antitumor immunity. Compared with conventional CAR-T cells and anti-PD-1-secreting CAR-T cells, data from in vitro and in vivo experiments showed that CAR-T cells with trap protein secretion further attenuated inhibitory T cell signaling, enhanced T cell persistence and expansion, and improved effector function and resistance to exhaustion. In the xenograft mouse model, CAR-T cells with trap protein secretion exhibited significantly enhanced antitumor immunity and efficacy. With these observations, we demonstrate the potential of trap protein self-secreting CAR-T cells as a potent therapy for solid tumors.

Abstract IA07: The QuEST for an effective immunotherapy for prostate cancer

Abstract Background: Immune checkpoint inhibitors have minimal activity in unselected CRPC patients (pts). Combination regimens that generate a tumor-directed immune response (vaccine) and facilitate the resulting anti-tumor immune activity (checkpoint blockade, cytokines) have shown synergy in preclinical models. BNVax is a therapeutic poxviral vaccine targeting brachyury, a transcription factor involved in invasion and metastasis. BA is a bifunctional fusion protein: anti-PD-L1 monoclonal antibody fused to the TGFb-β-RII receptor extracellular domain (a TGF-β trap). N-803 is an IL-15 superagonist complex. Here we report an interim efficacy analysis of the QuEST1 study, aimed to rapidly interrogate safety and efficacy of immunotherapy combinations in CRPC. Methods: Asymptomatic/minimally symptomatic CRPC pts received BNVax + BA (Arm 2.1) or BNVax + BA + N-803 (Arm 2.2). BNVax is given as 2 prime doses followed by boosts. BA 1,200 mg intravenously and N-803 15 µg/kg subcutaneously are given every 2 weeks. Efficacy is defined as objective response by RECIST v1.1 or PSA decrease ≥ 30% sustained for ≥ 21 days. Safety was a secondary endpoint. Results: As of 10/11/20, 26 CRPC pts enrolled and had 1 to 23 months follow up. 1/13 pts (Arm 2.1) had a PSA response sustained for 13 months. 5/12 (41.6%) pts on treatment for at least 12 weeks (Arm 2.2) had sustained PSA responses, including 2 pts who progressed on enzalutamide. Of 3 patients with measurable disease, 2 had confirmed PR by RECIST. Grade 3 TRAEs: 1 pancreatitis (Arm 2.1), 1 secondary adrenal insufficiency (Arm 2.1), 1 tumor hemorrhage (Arm 2.1), 1 hyperglycemia due to new onset diabetes mellitus (Arm 2.2), 1 cystitis (Arm 2.2), 1 Rash with eosinophilia (Arm 2.2). One patient had grade 4 neutropenia that resolved with growth factor support and oral corticosteroids. Conclusions: BVax + BA + N-803 demonstrated a manageable safety profile and preliminary evidence of efficacy in CRPC. Addition of N-803 in Arm 2.2 was associated with more activity in this small sample. Arm 2.2 met the predetermined threshold for efficacy and will expand (n=25). Citation Format: James L. Gulley. The QuEST for an effective immunotherapy for prostate cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr IA07.

Transforming growth factor β in breast cancer: another new trick for the old dog.

Two recent papers from the laboratory of Professor Ming Li demonstrate that inhibition of transforming growth factor β specifically in CD4+ T cells can suppress tumor growth through an unanticipated mechanism.

Cancer immunotherapy via targeted TGF-β signalling blockade in TH cells.

Cancer arises from malignant cells that exist in dynamic multilevel interactions with the host tissue. Cancer therapies aiming to directly kill cancer cells, including oncogene-targeted therapy and immune-checkpoint therapy that revives tumour-reactive cytotoxic T lymphocytes, are effective in some patients1,2, but acquired resistance frequently develops3,4. An alternative therapeutic strategy aims to rectify the host tissue pathology, including abnormalities in the vasculature that foster cancer progression5,6; however, neutralization of proangiogenic factors such as vascular endothelial growth factor A (VEGFA) has had limited clinical benefits7,8. Here, following the finding that transforming growth factor-β (TGF-β) suppresses T helper 2 (TH2)-cell-mediated cancer immunity9, we show that blocking TGF-β signalling in CD4+ T cells remodels the tumour microenvironment and restrains cancer progression. In a mouse model of breast cancer resistant to immune-checkpoint or anti-VEGF therapies10,11, inducible genetic deletion of the TGF-β receptor II (TGFBR2) in CD4+ T cells suppressed tumour growth. For pharmacological blockade, we engineered a bispecific receptor decoy by attaching the TGF-β-neutralizing TGFBR2 extracellular domain to ibalizumab, a non-immunosuppressive CD4 antibody12,13, andnamed it CD4 TGF-β Trap (4T-Trap). Compared with a non-targeted TGF-β-Trap, 4T-Trap selectively inhibited TH cell TGF-β signalling in tumour-draining lymph nodes, causing reorganization of tumour vasculature and cancer cell death, a process dependent on the TH2 cytokine interleukin-4 (IL-4). Notably, the 4T-Trap-induced tumour tissue hypoxia led to increased VEGFA expression. VEGF inhibition enhanced the starvation-triggered cancer cell death and amplified the antitumour effect of 4T-Trap. Thus, targeted TGF-β signalling blockade in helper T cells elicits an effective tissue-level cancer defence response that can provide a basis for therapies directed towards the cancer environment.

Arming Anti-EGFRvIII CAR-T With TGFβ Trap Improves Antitumor Efficacy in Glioma Mouse Models.

Glioblastoma (GBM) is an aggressive malignancy with poor prognosis. New therapeutic strategies for GBM are urgently needed. Although clinical studies have demonstrated the feasibility and safety of chimeric antigen receptor (CAR) T cell therapy for GBM, its efficacy has not been that impressive. The major limitation for anti-tumor efficacy of CAR-Ts is the immunosuppressive milieu of the GBM tumor microenvironment (TME). TGFβ, a substantial component in GBM, compromises the immune response and contributes to immune evasion and tumor progression. To overcome this limitation and improve the efficacy of CAR-T cells for GBM, we optimized an EGFRvIII-specific CAR construct with TGFRII ectodomain as a TGFβ-trap and generated TGFβ-resistant CAR-Ts for GBM therapy. We demonstrated that this TGFβ-trapped architecture enhanced anti-tumor efficacy of EGFRvIII-specific CAR-T and prolonged the survival of mice bearing GBM. In addition, the GBM-infiltrated microglia, typically considered tumorigenic, showed increased expression of M1 polarization markers after treatment with the TGFβ-trap CAR-Ts group, indicating that these microglia were polarized toward a pro-inflammatory and anti-tumorigenic phenotype. Overall, these results indicated that arming CAR-T cells with a TGFβ-trap diminishes the immunosuppressive effect and is a potential strategy to improve CAR-T efficacy for GBM therapy.

Abstract 1573: Association between TGF-β signaling and HMGA2, a potential biomarker for bintrafusp alfa in triple negative breast cancer

Abstract 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.

AVID200, a Potent Trap for TGF-β Ligands Inhibits TGF-β1 Signaling in Human Myelofibrosis

Myelofibrosis (MF) is caused by driver mutations which upregulate JAK/STAT signaling. The only curative treatment for MF is hematopoietic stem cell transplant. Ruxolitinib alleviates many of the symptoms in MF but does not significantly alter survival. There is, therefore, an urgent need for additional rational therapies for MF. Bone marrow fibrosis and collagen deposition are hallmarks of MF which have been attributed to megakaryocyte (MK) derived TGFβ, which also plays a role in myelo-proliferation. There are three isoforms of TGFβ (TGFβ1, β2, and β3). AVID200, which was constructed by fusing TGFβR ectodomains to IgG Fc regions, is a potent TGFβ trap with pM potency against two of the three TGFβ ligands, TGFβ1 and β3 (IC50 values of ~ 3 pM ). AVID200's IC50 for TGFβ2 is ~4,000-fold higher indicating that it has minimal activity against TGFβ2, which is desirable since TGFβ2 is a positive regulator of hematopoiesis.We explored the therapeutic potential of AVID200 by culturing MF or normal donor (ND) mononuclear cells (MNCs) first in the presence of stem cell factor and thrombopoietin (TPO) and then TPO alone in order to generate MK-enriched populations. Although the percentage of mature MKs from ND and MF MNCs was similar, the absolute number of CD41+/CD42+ MKs generated from MF MNCs was two-fold greater than ND MNCs.To determine the levels of TGFβ secreted by the MKs we screened MF and ND MNC conditioned media (CM). We observed significantly higher levels of TGFβ1 but not TGFβ2 and TGFβ3 in MF MK CM. The effects of AVID200 on MKs were then evaluated by measuring the levels of phosphorylated SMAD2. Treatment with 0.001 - 0.1 nM AVID200 decreased phosphorylation of SMAD2, suggesting that AVID200 blocks autocrine MK TGFβ signaling.The increased levels of TGFβ in MF patients promote the proliferation and deposition of collagen by mesenchymal stem cells (MSCs). Cellular proliferation of MSCs was evaluated following treatment with either recombinant TGFβ1 or ND/MF CM in the presence or absence of AVID200. In the absence of AVID200, both recombinant TGFβ1 and MK-derived CM increased the proliferation of MSCs by 1.4- and 1.6-fold respectively, which returned to basal levels with the addition of increasing concentrations of AVID200. These data indicate that AVID200 directly blocks the effect of TGFβ1 on MSCs.MF stroma is characterized by an increase in Type I collagen. We therefore examined if treatment with AVID200 interferes with the ability of TGFβ1 to induce collagen expression by MSCs. MSCs were cultured in presence of recombinant TGFβ1 alone or in combination with varying concentrations of AVID200 for 72 hours. Recombinant TGFβ1 alone induced an increase in COL1A1 mRNA expression as compared to untreated controls (p<0.01). Addition of AVID200 eliminated the TGFβ-mediated increase in COL1A1 expression in a dose dependent manner. ND and MF MK-derived CM also increased COL1A1 expression by MSCs as compared to un-treated controls (p<0.01) and that effect was eliminated by AVID200 treatment (p<0.01). We next demonstrated that TGFβ1 activated pSMAD2 in MSCs without affecting total SMAD2/3 expression and that SMAD2 phosphorylation was reduced by adding AVID200. Furthermore, AVID200 treatment decreased pSTAT3 which is associated with the ability of TGFβ to induce fibrosis.We next investigated the effect of AVID200 on MF hematopoiesis. Briefly, MNCs (which produce TGFβ) from two JAK2V617F+ MF patients were incubated with or without 50 nM of AVID200 and plated in semi-solid media. Treatment with AVID200 did not affect the overall number of colonies generated, but reduced the numbers of JAKV617F+ colonies while increasing the numbers of WT colonies: for PT1, there were 32% JAKV617F+ CFUs in untreated cultures (11 JAKV617F+/34 total colonies) versus 16% JAKV617F+ CFUs (7 JAKV617F+/42 total CFUs) in AVID200 treated cultures; for PT2 there were 100% JAKV617F+ CFUs in untreated cultures (37 JAKV617F+/37 total CFUs) versus 94% JAKV617F+ CFUs (49 JAK2V617F+/52 total CFUs) in AVID200 treated cultures. The in vivo effects of AVID200 on the development of MF in GATA1low mice will be presented at the meeting.These data indicate that AVID200 selectively suppresses TGFβ1 signaling associated with the proliferation of MSCs and type I collagen synthesis, and depletes MF MNCs of JAK2V617F+progenitor cells. We conclude that AVID200 is a promising agent for treating MF patients which will be evaluated in a phase 1 clinical trial. DisclosuresMascarenhas:Novartis: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merck: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding. Iancu-Rubin:Incyte: Research Funding; Merck: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Summer Road: Research Funding; Merus: Research Funding; Janssen: Research Funding; Formation Biologics: Research Funding.

Abstract 1759: AVID200, a highly potent TGF-beta trap, exhibits optimal isoform selectivity for enhancing anti-tumor T-cell activity, without promoting metastasis or cardiotoxicity

Abstract The purpose of the studies presented here is to examine the ability of the novel TGF-β-neutralizing agent, AVID200, to reverse immunosuppression in the tumor microenvironment, and to determine the safety profile of AVID200 in non-human primates. TGF-β is a secreted protein that is aberrantly produced by tumors, and which promotes cancer progression primarily by suppressing both the innate and adaptive immune systems. AVID200 is a computationally-designed, avidity-enhanced, receptor ectodomain-based trap that binds and neutralizes TGF-β1 and -β3. We demonstrate, in tumor cell-based assays, that AVID200 potently neutralizes TGF-β1 and -β3 with low pM potency. Using a syngeneic 4T1 triple negative breast cancer (TNBC) model, we report that AVID200, in a dose-dependent manner, enhances the capacity of T-cells isolated from draining lymph nodes to specifically recognize and kill 4T1 tumor cells. The anti-tumor T-cell-activating potency of AVID200 was observed to be higher than that of the pan-neutralizing TGF-β antibody, 1D11. AVID200 was designed to have minimal activity against TGF-β2. This isoform specificity was chosen since it has been reported that inhibition of the TGF-β2 isoform can promote metastasis. We report that AVID200 does not increase the number of foci in a metastasis assay, whereas pan TGF-β neutralizing agents do. This supports the concept that blockade of TGF-β2 is undesirable. Also, it has been reported that pan-TGF-β blockers can exhibit cardiac toxicity in animal models and in humans. It has been proposed that this is due to TGF-β2 neutralization since this is the main isoform implicated in normal cardiac function. To determine if AVID200 exhibits cardiotoxicity, we tested AVID200 in a pilot non-human primate study. No adverse cardiac events were observed at doses of up to 30 mg/kg. An update on GLP toxicology studies will be presented at the meeting. In conclusion, AVID200 is a novel TGF-β trap that potently blocks TGF-β-1 and -3 isoforms, which results in reversal of immunosuppression in the tumor microenvironment. It has minimal activity against TGF-β2 and accordingly does not detectably promote cardiac toxicity and metastasis. Thus, AVID200 is a promising new immunotherapy that selectively inhibits the TGF-β1 and -3 isoforms, thereby enhancing desirable anti-tumor immunity, while avoiding the tumor-promoting and cardiotoxic effects resulting from TGF-β2 neutralization. Citation Format: Maureen D. O'Connor-McCourt, Gilles Tremblay, Anne Lenferink, Traian Sulea, John Zwaagstra, James Koropatnick. AVID200, a highly potent TGF-beta trap, exhibits optimal isoform selectivity for enhancing anti-tumor T-cell activity, without promoting metastasis or cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1759.

A Novel TGFβ Trap Blocks Chemotherapeutics-Induced TGFβ1 Signaling and Enhances Their Anticancer Activity in Gynecologic Cancers.

Purpose: We investigated the mechanisms of how TGFβ pathway is activated by chemotherapeutics and whether a novel TGFβ trap called RER can block chemotherapeutics-induced TGFβ pathway activation and enhance their antitumor activity in gynecologic cancer.Patients and Methods: An unbiased bioinformatic analysis of differentially expressed genes in 31 ovarian cases due to chemotherapy was used to identify altered master regulators. Phosphorylated Smad2 was determined in 30 paired cervical cancer using IHC. Furthermore, the effects of chemotherapeutics on TGFβ signaling and function, and the effects of RER on chemotherapy-induced TGFβ signaling were determined in gynecologic cancer cells.Results: Chemotherapy-induced transcriptome alteration in ovarian cancer was significantly associated with TGFβ signaling activation. Chemotherapy was found to activate TGFβ signaling as indicated by phosphorylated Smad2 in paired cervical tumor samples (pre- and post-chemotherapy). Similar to TGFβ1, chemotherapeutics were found to stimulate Smad2/3 phosphorylation, cell migration, and markers related to epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC). These TGFβ-like effects were due to the stimulation of TGFβ1 expression and secretion, and could all be abrogated by TGFβ inhibitors including a novel TGFβ trap protein called RER both in vitro and in vivo Importantly, combination treatment with RER and cisplatin showed a higher tumor inhibitory activity than either agent alone in a xenograft model of ovarian cancer.Conclusions: Chemotherapeutics can stimulate TGFβ1 production and consequently enhance TGFβ signaling, EMT, and CSC features resulting in reduced chemo-sensitivity. Combination therapy with a TGFβ inhibitor should alleviate this unintended side effect of chemotherapeutics and enhance their therapeutic efficacy. Clin Cancer Res; 24(12); 2780-93. ©2018 AACR.

M7824, a novel bifunctional anti-PD-L1/TGFβ Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine

ABSTRACTTumors evade host immune surveillance through multiple mechanisms, including the generation of a tumor microenvironment that suppresses immune effector function. Secretion of TGFβ and upregulation of immune checkpoint programmed cell death ligand-1 (PD-L1) are two main contributors to immune evasion and tumor progression. Here, we examined the efficacy of a first-in-class bifunctional checkpoint inhibitor, the fusion protein M7824, comprising the extracellular domain of human TGFβRII (TGFβ Trap) linked to the C-terminus of human anti-PD-L1 heavy chain (αPD-L1). We demonstrate that M7824 reduces plasma TGFβ1, binds to PD-L1 in the tumor, and decreases TGFβ-induced signaling in the tumor microenvironment in mice. In murine breast and colon carcinoma models, M7824 decreased tumor burden and increased overall survival as compared to targeting TGFβ alone. M7824 treatment promoted CD8+ T cell and NK cell activation, and both of these immune populations were required for optimal M7824-mediated tumor control. M7824 was superior to TGFβ- or αPD-L1-targeted therapies when in combination with a therapeutic cancer vaccine. These findings demonstrate the value of using M7824 to simultaneously target TGFβ and PD-L1/PD-1 immunosuppressive pathways to promote anti-tumor responses and efficacy. The studies also support the potential clinical use of M7824 as a monotherapy or in combination with other immunotherapies, such as therapeutic cancer vaccines, including for patients who have progressed on αPD-L1/αPD-1 checkpoint blockade therapies.

Connective tissue growth factor expression after angiotensin II exposure is dependent on transforming growth factor-β signaling via the canonical Smad-dependent pathway in hypertensive induced myocardial fibrosis.

Introduction:Transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) are often described as the initial pro-fibrotic mediators upregulated early in fibrosis models dependent on angiotensin II (Ang-II). In the present study, we explore the mechanistic link between TGF-β and CTGF expression by using a novel TGF-β trap.Materials and methods:NIH/3T3 fibroblasts were subjected to TGF-β with or without TGF-β trap or 1D11 antibody, CTGF or CTGF plus TGF-β for six or 24 hours, and then used for quantitative real-time polymerase chain reaction (qRT-PCR) or immunocytochemistry. Male C57BL/6 mice were infused with Ang-II and randomly assigned TGF-β trap for six or 24 hours. Hearts were harvested for histological analyses, qRT-PCR and western blotting.Results:Exogenous TGF-β-induced fibroblasts resulted in significant upregulation of CTGF, TGF-β and type I collagen transcript levels in vitro. Additionally, TGF-β promoted the differentiation of fibroblasts into α-SMA+ myofibroblasts. CTGF expression was reduced by the addition of TGF-β trap or neutralizing antibody, confirming that its expression is dependent on TGF-β signaling. In contrast, exogenous CTGF did not appear to have an effect on fibroblast production of pro-fibrotic transcripts or fibroblast differentiation. Ang-II infusion in vivo led to a significant increase in TGF-β and CTGF mRNA expression at six and 24 hours with corresponding changes in Smad2 phosphorylation (pSmad2), indicative of increased TGF-β signaling. Ang-II animals that received the TGF-β trap demonstrated reduced CTGF mRNA levels and pSmad2 at six hours, suggesting that early CTGF expression is dependent on TGF-β signaling.Conclusions:We demonstrated that CTGF expression is dependent on TGF-β signaling both in vitro and in vivo in a model of myocardial fibrosis. This also suggests that early myocardial CTGF mRNA expression (six hours) after Ang-II exposure is likely dependent on latent TGF-β activation via the canonical Smad-dependent pathway in resident cardiac cells.