BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PAs) and progressive increase in pulmonary vascular resistance leading to right ventricular failure. Although several drugs are approved for the treatment of PAH, mortality rates remain high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. METHODS: The expression of the RGD (arginylglycylaspartic acid)–binding integrin α5β1 was assessed in PAs, PA smooth muscle cells, and PA endothelial cells from patients with PAH and controls using NanoString, immunoblotting, and Mesoscale Discovery assays. RNA sequencing was conducted to identify gene networks regulated by α5β1 inhibition in PAH PA smooth muscle cells. The therapeutic efficacy of α5β1 inhibition was evaluated using a novel small molecule inhibitor and selective neutralizing antibodies in Sugen/hypoxia and monocrotaline rat models, with validation by an external contract research organization. Comparisons were made against standard-of-care therapies (ie, macitentan, tadalafil) and sotatercept and efficacy was assessed using echocardiographic, hemodynamic, and histological assessments. Ex vivo studies using human precision-cut lung slices were performed to further assess the effects of α5β1 inhibition on pulmonary vascular remodeling. RESULTS: We found that the arginine-glycine-aspartate RGD-binding integrin α5β1 is upregulated in PA endothelial cells and PA smooth muscle cells from patients with PAH and remodeled PAs from animal models. Blockade of the integrin α5β1 or depletion of the α5 subunit downregulated FOXM1 (forkhead box protein M1)–regulated gene networks, resulting in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. We demonstrated that α5β1 integrin blockade safely attenuates pulmonary vascular remodeling and improves hemodynamics and right ventricular function and matched or exceeded the efficacy of standard of care and sotatercept in multiple preclinical models. Ex vivo studies further validated its potential in reversing advanced remodeling in human precision-cut lung slices. CONCLUSIONS: These findings establish α5β1 integrin as a pivotal driver of PAH pathology and we propose its inhibition as a novel, safe, and effective therapeutic strategy for PAH.

Inhibition of integrin αvβ6 is a promising approach to the treatment of fibrotic disease such as idiopathic pulmonary fibrosis. Screening a small library combining head groups that stabilize the bent-closed conformation of integrin αIIbβ3 with αv integrin binding motifs resulted in the identification of hit compounds that bind the bent-closed conformation of αvβ6. Crystal structures of these compounds bound to αvβ6 and related integrins revealed opportunities to increase potency and selectivity, and these efforts were accelerated using accurate free energy perturbation (FEP+) calculations. Optimization of PK parameters including permeability, bioavailability, clearance, and half-life resulted in the discovery of development candidate MORF-627, a highly selective inhibitor of αvβ6 that stabilizes the bent-closed conformation and has good oral PK. Unfortunately, the compound showed toxicity in a 28-day NHP safety study, precluding further development. Nevertheless, MORF-627 is a useful tool compound for studying the biology of integrin αvβ6.

Introduction: Pulmonary arterial hypertension (PAH) is marked by pathological remodeling of distal arteries, driven by hyperplasia of pulmonary arterial smooth muscle cells (PASMCs), dysfunction of pulmonary arterial endothelial cells (PAECs), and increased deposition of the extracellular matrix (ECM). Hypothesis: Considering the crucial role of ECM in vascular restructuring, we investigated the potential of therapeutically targeting a5b1, a fibronectin-binding integrin involved in cell proliferation and angiogenesis, for PAH treatment. Aim: To evaluate the therapeutic potential of targeting a5b1 for the treatment of PAH. Methods: We have developed potent, orally bioavailable small molecule inhibitors (SMis) and monoclonal antibodies (mAbs) targeting a5b1 to evaluate its therapeutic potential. The effect of a5b1 inhibition was assessed in cultured PASMCs, human precision-cut lung slices (PCLS), and a rat Sugen/hypoxia PAH model. Results: PAH patients exhibited increased expression of fibronectin and a5b1 in distal pulmonary arteries. Selective inhibition of a5b1 in cultured PASMCs modulated multiple pathways involved in cell cycle regulation at both transcriptional and post-transcriptional levels, thereby blocking cellular proliferation. PCLS treated with a5b1 inhibitors showed decreased expression of pathways involved in ECM deposition and reduced levels of smooth muscle cell markers. In the rat Sugen/hypoxia PAH model, a5b1 inhibition with either an SMi or mAb significantly improved cardiac and vascular function by reducing pulmonary arterial wall thickness, right ventricular hypertrophy, and fibrosis. Cardiac improvement is further evidenced by a reduction in circulating NT-proBNP and NT-proANP levels. Conclusion: These findings reveal a previously underappreciated role for a5b1 in PAH pathogenesis and support the potential of a5b1 inhibition as a disease-modifying therapeutic strategy for managing PAH.

Introduction: Right ventricular (RV) function is an important prognosis factor in pulmonary arterial hypertension (PAH). In response to pressure overload, the RV undergoes morphological changes driven by the accumulation of extracellular matrix (ECM) elements and cardiomyocyte hypertrophy. Integrins (Itg) are members of the cell adhesion receptor superfamily. Through ECM-binding, Itg are known to transduce extracellular cues to cardiac cells resulting in the activation of pro-hypertrophic and pro-fibrotic signaling pathways. Among them, integrin α5β1 is one of the most abundant heterodimers in cardiac tissue. We thus hypothesized that α5β1 signaling promotes pathological RV remodeling. Methods&Results: Using Western blot (WB), we found elevated levels of Itgα5 and Itgβ1 protein in RV isolated from PAH patients compared to controls. Similar findings were observed in monocrotaline (MCT) and pulmonary artery banding (PAB) rats. In adult rat cardiomyocytes, pharmacological inhibition of α5β1 decreased phenylephrine-induced hypertrophy and reversed established hypertrophy induced by MCT treatment (assessment of cell surface area by F-actin labeling). In human RV fibroblasts (RVFB) inhibition of α5β1 prevented TGFβ1-induced activation of control RVFB and decreased proliferation and activation of RVFB derived from PAH patients (WB PCNA, αSMA, COL1; immunofluorescence Ki67). In vivo , inhibition of α5β1 using a small molecule inhibitor or a blocking-antibody improved RV dysfunction (CO, TAPSE, RVFAC, RV strain, as assessed by echocardiography, right heart catheterization and cardiac magnetic resonance imaging) and diminished RV hypertrophy and fibrosis (Fulton index, H&E and Masson’s trichrome staining) in MCT-injected rats with established PAH and PAB rats. Ex vivo , inhibition of α5β1 attenuated cardiomyocyte hypertrophy and RV fibrosis in human precision-cut RV slices exposed to phenylephrine and TGFβ1. Conclusion: Our results suggest that integrin α5β1 plays a key role in pathological RV remodeling in PAH.

Introduction: Pulmonary arterial hypertension (PAH) is characterized by progressive obstruction and decreased compliance of pulmonary arteries (PA), leading to right ventricular failure and premature death. Sustained proliferation and resistance to apoptosis of PAs smooth muscle and endothelial cells (PASMCs, PAECs) and accumulation of extracellular matrix (ECM) elements are key features contributing to vascular remodeling. Integrins, members of the cell adhesion receptor superfamily, are known to promote cell proliferation and survival through ECM binding. Despite the recognition of the role of ECM elements in vascular remodeling in PAH, the contribution of integrins remains poorly studied. We hypothesized that integrin signaling could promote PAH-PASMCs and PAH-PAECs proliferation and resistance to apoptosis contributing to vascular remodeling. Methods&Results: Using Western blot (WB) and Meso Scale Discovery, we found that α5β1 was upregulated in distal PAs, PA smooth muscle cells (PASMC) and PA endothelial cells (PAEC) from PAH patients compared to controls. Small molecule, antibody (M200), or siRNA-mediated inhibition of α5β1 decreased PAH-PASMC and PAH-PAEC proliferation (WB, MCM2 and Ki67 labeling), resistance to apoptosis (WB, Survivin and Annexin V labeling) and migration (scratch test assay). RNA sequencing analysis revealed that inhibition of α5β1 regulates a FOXM1-dependant gene network through the activation of focal adhesion kinase, resulting in mitotic defects (Immunofluorescence: α-tubulin and pericentrin labeling). In vivo , in both monocrotaline and Sugen/hypoxia rats with established PAH, inhibition of α5β1 using a small molecule inhibitor or a neutralizing antibody alone or in combination with macitentan and tadalafil improved hemodynamics (RHC and echocardiography: mean pulmonary arterial pressure, cardiac output, pulmonary artery acceleration time) and vascular remodeling (Elastica Van Gieson staining). Ex vivo , inhibition of α5β1 decreased vascular remodeling in human precision-cut lung slices (PCLS) from PAH patients and attenuated growth factors-induced vascular remodeling in control PCLS. Conclusion: Integrin α5β1 plays a key role in vascular remodeling in PAH and represents a novel therapeutic target.

Abstract Objective Vedolizumab has been shown to reduce the antibody response to an oral cholera, but not intramuscular hepatitis B vaccine in healthy subjects (Wyant, Gut 2015). The generation of an immune response to an oral antigen (Ag) begins at inductive sites such as Peyer’s patches (PP) followed by cell migration to effector sites in the gut. The integrin α4β7 is known to play a major role in cell trafficking to gut-associated lymphoid tissues. This study assessed whether MT-102, a potent and selective small molecule α4β7 inhibitor has similar effects on immunizations to those reported in the clinic for vedolizumab. Methods An in vivo immunization model system was established to determine the effects of MT-102, dosed via osmotic minipump, on the development of a gut-specific immune response. Mice were orally immunized with cholera toxin (CTX), and anti-CTX antibodies were quantified in fecal and plasma samples. The impact of MT-102 on lymphocytes within the PP of immunized mice was determined by flow cytometry. Results MT-102 delayed the production of anti-CTX IgA and IgG antibodies as measured in fecal samples without perturbing total gut IgA levels. Furthermore, MT-102 also inhibited the levels of Ag-specific antibodies in the plasma following repeated CTX oral immunizations. Blocking α4β7 induced a decrease in the frequency of B cells in the PP of treated mice compared to the control group. Conclusion α4β7-specific inhibitor, MT-102, effectively diminished the mucosal antibody response to an orally delivered Ag in mice. These results are consistent with what has been reported clinically in vedolizumab-treated subjects and thus establishes that potent small molecule inhibitors of α4β7 can replicate the pharmacology of an antibody.

Background Ovarian cancer (OC) is the most lethal gynecologic malignancy. Despite the initial high response rate to chemotherapy, most patients relapse. Immunotherapy offers potential for long-term remission but single checkpoint inhibition benefits less than 15% of patients. Growing evidence suggests that immune-checkpoint blockade (ICB) is enhanced when combined with therapies that target tumor tolerance. Transforming growth factor beta (TGF-β) is associated with resistance to immunotherapy and tumor tolerance. Integrin αvβ8 controls cell-type-specific activation of TGF-β and αvβ8 antagonism promotes anti-tumor immunity leading to tumor regression in ICB refractory tumors.1,2 We explored the impact of αvβ8 inhibition to restore ICB response in a murine ovarian carcinoma model and performed blood cytokine profiling to search for pharmacodynamic markers of response to treatment. Methods Bioinformatic analysis on bulk and single-cell levels of public OC datasets was performed to evaluate ITGB8 and TGF-β-related gene signatures. qPCR was used for the detection of TGF-β and ITGB8 expression in ID8, a murine ovarian carcinoma model (ID8-Luc-mCh-Puro.TD1). ID8 tumors unresponsive to PD-1/L1 inhibition were established to mimic advanced-stage disease. The efficacy of αvβ8 mAbs in combination with PD-L1 blockade was evaluated. Mice were evaluated for survival for 65 days. Blood samples were harvested before treatment and on days 1,8 and 14. Plasma was analyzed via Luminex for a total of 30 ­­­cytokines. Transcriptomic analyses of MC38 and EMT6 mouse tumors were performed by bulk RNA seq. Results In high-grade serous OC, ITGB8 expression is associated with shorter overall survival and increased gene signatures of TGF-β1/3 pathways. The ID8 murine ovarian carcinoma model is unresponsive to PD-1/L1 inhibition and expresses Itgb8 together with TGF-β 1/3. The combination of αvβ8 and PD-L1 mAbs led to complete tumor regression in 9/10 mice relative to 0/10 in the PD-L1 group, resulting in superior survival (P=0.0001) (Fig 1 A, B). The combination therapy led to upregulation of blood granzyme B (P=0.05), IL-27(P=0.03), and IL-1a (P= 0.008) at 14-day post-treatment and transient upregulation of CCL3 (P<0.0001) and CCL7 (P= 0.0005) at 8-day post-treatment. Cross-analysis of tumor tissues from the colon and breast syngeneic models after treatment showed similar transcript upregulation for cytokines. Conclusions Inhibition of αvβ8 renders advanced ID8 tumors sensitive to immune checkpoint therapy, leading to tumor eradication and superior survival. These data provide evidence that a treatment modality targeting αvβ8-mediated TGF-β 1/3 pathways may enhance patients' sensitivity to checkpoint therapies. Plasma blood-based biomarkers serve as a non-invasive method for response assessment to αvβ8-based therapy. References Reszka-Blanco NJ, Yadav V, Krumpoch M, Cappellucci L, Cui D, Dowling JE, et al. Inhibition of integrin avß8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer. AACR; Cancer Res 2021;81(13_Suppl): Abstract nr 1559 Dodagatta-Marri E, Ma H-Y, Liang B, Li J, Meyer DS, Chen S-Y, et al. Integrin avß8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy. Cell Report. 2021; 36(1): 109309

Abstract Introduction We explored whether integrin αvβ8 inhibition potentiates immune checkpoint blockade (ICB) in syngeneic orthotopic models of breast cancer. Integrin αvβ8 mediates cell type specific and tissue localized activation of TGFβ1/3 to regulate the immune system. For example, αvβ8 expressed on dendritic cells (DC) in the intestine has been shown to be a key mediator of tolerance, maintaining gut immunologic homeostasis. Methods Efficacy was evaluated in combination with anti-PD-1 in EMT6 and PyMT breast cancer syngeneic mouse models. A potent αvβ8 inhibitor was orally administered at 60mg/kg BID for 21 days. Anti-αvβ8 or non-isoform specific anti-TGFβ mAbs were dosed TIWx3 at 7 and 10mg/kg, respectively. Tumor volumes are presented as mean±SEM. Statistics were performed by t test, one-way ANOVA, or log-rank test. Flow cytometry and transcriptome analysis on bulk and single-cell levels were used to assess the mechanism of action in EMT6. Results A similar αvβ8 expression pattern on DC, macrophages and regulatory T cells (Treg) was observed in mouse models and human tumors. Combination of oral αvβ8 inhibitor with anti-PD-1 was efficacious in the primary ICB resistant EMT6 model and resulted in superior tumor regression during treatment (p=0.0003) and improved survival with 5/12 complete responders relative to 0/12 in anti-PD-1 alone. Across studies the αvβ8 inhibitor phenocopied the results obtained with αvβ8 and TGFβ mAbs (n=3 independent studies). Complete responders re-challenged 89 days after treatment with EMT6 or 4T1 tumors showed no EMT6 tumor growth, suggesting the combination induced long-term immunologic memory. Analysis of tumors by flow cytometry showed combination resulted in increased CD8 T cell infiltrates (p=0.0006), T cell activation (CD8+CD69+, p=0.0194) and IFN-γ expression (CD8+IFN-γ+, p=0.0021). Single cell transcriptomic analysis of lymph nodes showed that αvβ8 inhibition potentiated DC co-stimulation (Cd40, Cd83/6) and migration (Ccr7, Cxcl16, Ccl22). Moreover, combination treatment led to tumor infiltrated Treg dysfunction including downregulation of Ctla4, Il10 and Tigit, and upregulation of Ifn-γ. The observation of these anti-tolerance and pro-inflammatory signatures in DC and Treg has not been described previously. Anti-tumor efficacy was driven by immune-mediated mechanisms as confirmed by a CD8 depletion study. Efficacy was confirmed in PyMT breast cancer model. Conclusions An αvβ8 inhibitor in combination with anti-PD-1 showed efficacy in syngeneic mouse models, supported by increased T cell infiltrates and evidence of reduced tumor tolerance. These results show that an orally administered αvβ8 targeted inhibitor is a potent modulator of anti-tumor immune response acting across the immunologic synapse, and is a promising therapeutic approach to ICB refractory tumors. Citation Format: Natalia J. Reszka-Blanco, Vinod Yadav, Megan Krumpoch, Laura Cappellucci, Dan Cui, James E. Dowling, Elizabeth Gwara, Bryce Harrison, Dooyoung Lee, Fu-Yang Lin, Lia Luus, Meghan Monroy, Terence I. Moy, Eugene Nebelitsky, Qi Qiao, Andrew Sullivan, Dawn Troast, Blaise Lippa, Bruce Rogers, Adrian S. Ray. Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer [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 1559.