Inhibition of RANKL and Sema4d Improves Residual Ridge Resorption in Mice.

Residual ridge resorption (RRR) is a chronic and progressive bone resorption following tooth loss. It causes deterioration of the oral environments and leads to the pathogenesis of various systemic diseases. However, the molecular mechanisms and risk factors for RRR progression are still unclear and controversial. In this study, we developed a tooth extraction model using mice for analyzing long-term morphological and gene expression changes in the alveolar bone. We further applied ovariectomy to this model to elucidate the effects of osteoporosis on RRR progression. As a result, the alveolar bone loss was biphasic and consisted of rapid loss in the early stages and subsequently slow and sustained bone loss over a long period. Gene expression analysis indicated that ovariectomy increased the expression of pro-inflammatory cytokines in the alveolar bone and prolonged the activation of osteoclasts same as histological analysis. Furthermore, the expressions of Tnfsf11 and Sema4d kept increasing for a long time in OVX mice. Administration of neutralization antibodies for receptor activator of NF-κB ligand (RANKL) effectively suppressed RRR. Similarly, inhibition of Semaphorin 4d (Sema4d) also improved alveolar bone loss. This study demonstrated that osteoporosis is a risk factor for RRR and that RANKL and Sema4d suppression are potential treatments.

Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.

434 Phase 1/2 study to evaluate pepinemab in combination with pembrolizumab in advanced, recurrent or metastatic head and neck cancer (KEYNOTE B84)

BackgroundImmunosuppressive myeloid cells in the tumor microenvironment (TME) are a critical limitation to the efficacy of immune checkpoint inhibitors (ICIs) in patients with head and neck squamous cell carcinoma (HNSCC). Both semaphorin 4D (SEMA4D, CD100) and MDSCs are reported to play important roles in the growth and progression of HNSCC. Preclinical and clinical data demonstrated that antibody blockade of SEMA4D promotes tumor infiltration and activation of dendritic cells and CD8+ T cell, reverses immunosuppression, including attenuation of MDSC recruitment and function, and leading to enhanced efficacy of ICIs.1 2 In a study evaluating pepinemab, a humanized SEMA4D blocking antibody, in combination with avelumab in patients with non-small cell lung cancer, the combination appeared to provide clinical benefit in patients with difficult to treat ICI-resistant and PD-L1-low tumors.3 Pembrolizumab is approved as first line therapy as monotherapy or in combination with chemotherapy in recurrent or metastatic (R/M) HNSCC, however not all patients respond to ICIs and require more effective treatments.MethodsKEYNOTE B84 (NCT04815720) is a multicenter, single-arm open-label study to evaluate the safety, efficacy, PK/PD of pepinemab in combination with pembrolizumab in subjects with locally advanced, R/M HNSCC. Subjects with measurable disease per RECIST1.1 will be enrolled, including oropharynx, oral cavity, hypopharynx and larynx, and ECOG PS of 0 or 1. Subjects who have received prior ICIs are excluded. This study will include a Safety Run-in phase (n=3–18) and a Dose Expansion (maximum n=62) phase. Pepinemab, which is well-tolerated in combination with other ICIs, will be evaluated starting with the highest intended dose of 20 mg/kg, in combination with 200 mg pembrolizumab, both administered intravenously every 3 weeks. The Dose Expansion phase will include an even distribution of subjects who have combined positive scores of <20 and ≥20. The primary efficacy endpoint is ORR, and secondary endpoints include DOR, OS, PFS, as well as exploratory biomarker analysis. Pre- and on-treatment biopsies will be collected for evaluation of immune contexture in TME.ResultsScreening has been initiated at several of a planned total of 18 sites. Multiplex immunohistochemistry (IHC) panels have been established to phenotype cells in the TME, including CD8+ T cells, DCs, MDSCs, Tregs, monocytes, macrophages.ConclusionsThere remains a clear unmet need for more effective immunomodulatory treatment options to overcome immunosuppressive factors in the TME. The KEYNOTE B84 study will evaluate pepinemab as a potential treatment option to overcome resistance to and enhance activity of pembrolizumab in HNSCC.Trial RegistrationNCT04815720ReferencesClavijo PE, Friedman J, Robbins Y, Moore EC, Smith E, Zauderer M, Evans EE, Allen CT. Semaphorin4D Inhibition Improves Response to Immune-Checkpoint Blockade via Attenuation of MDSC Recruitment and Function. Cancer Immunol Res 2019 Feb;7(2):282–291.Evans EE, Jonason AS Jr, Bussler H, Torno S, Veeraraghavan J, Reilly C, Doherty MA, Seils J, Winter LA, Mallow C, Kirk R, Howell A, Giralico S, Scrivens M, Klimatcheva K, Fisher TL, Bowers WJ, Paris M, Smith ES, Zauderer M. Antibody Blockade of Semaphorin 4D Promotes Immune Infiltration into Tumor and Enhances Response to Other Immunomodulatory Therapies. Cancer Immunol Res 2015 Jun;3(6):689–701.Shafique MR, Fisher TL, Evans EE, Leonard JE, Pastore DRE, Mallow CL, Smith E, Mishra V, Schröder A, Chin KM, Beck JT, Baumgart MA, Govindan R, Gabrail NY, Spira AI, Seetharamu N, Lou Y, Mansfield AS, Sanborn RE, Goldman JW, Zauderer M. A Phase Ib/II Study of Pepinemab in Combination with Avelumab in Advanced Non-Small Cell Lung Cancer. Clin Cancer Res 2021 Jul 1;27(13):3630–3640.Ethics ApprovalThis study was approved by WIRB Copernicus Group’s Ethics Board on 11Feb2021; approval number 20210250.

Tenofovir Modulates Semaphorin 4D Signaling and Regulates Bone Homeostasis, Which Can Be Counteracted by Dipyridamole and Adenosine A2A Receptor

Semaphorin 4D (Sema4D) is a neurotrophin that is secreted by osteoclasts and binds to its receptor PlexinB1 on osteoblasts to inhibit their differentiation and function. Adenosine A2A activation inhibits osteoclast Sema4D-mediated secretion, diminishes inflammatory osteolysis and prevents bone loss following tenofovir (one of the most used antivirals in HIV). Therefore, tenofovir might activate Sema4D signaling to alter bone turnover. Female C57Bl/6/A2AKO mice were ovariectomized and treated with saline (control), tenofovir 75 mg/Kg/day, dipyridamole 25 mg/Kg/day or a combination for 5 weeks and long bones were prepared for histology. Primary murine-induced osteoclast/osteoblast were challenged with tenofovir/dipyridamole 1 μM each, and the expression of Sema4D/PlexinB1, RhoA/ROCK/IGF1R was studied by RT-PCR, Western blot and immunostaining. In vivo tenofovir showed an increased expression of Sema4D when compared to control mice, and dipyridamole reverted the expression in an A2A-dependent manner. In vitro, tenofovir increases Sema4D expression and secretion in osteoclast precursors, and pre-treatment with dipyridamole reverted this effect. pRhoA and ROCK1 activation were increased and IRS1/IGF1R expression was diminished by tenofovir in the Vav3/ARHGAP18 mechanism in osteoblast precursors and reverted by dipyridamole in an A2A-dependent manner. This suggests that tenofovir increases bone loss by activation of Sema4D/PlexinB1 signaling, which inhibits osteoblast differentiation. Agents that increase local adenosine concentrations, such as dipyridamole, might prevent bone loss following the inhibition of this pathway.

Anti-Semaphorin 4D Rescues Motor, Cognitive and Respiratory Phenotypes in a Rett Syndrome Mouse Model

Rett syndrome is a neurodevelopmental disorder caused by mutations of the methyl-CpG binding protein 2 gene. Abnormal physiological functions of glial cells contribute to pathogenesis of Rett syndrome. Semaphorin 4D (SEMA4D) regulates processes central to neuroinflammation and neurodegeneration including cytoskeletal structures required for process extension, communication, and migration of glial cells. Blocking SEMA4D-induced gliosis may preserve normal glial and neuronal function and rescue neurological dysfunction in Rett syndrome. We evaluated the pre-clinical therapeutic efficacy of an anti-SEMA4D monoclonal antibody in the Rett syndrome Mecp2T158A transgenic mouse model and investigated the contribution of glial cells as a proposed mechanism of action in treated mice and in primary glial cultures isolated from Mecp2T158A/y mutant mice. SEMA4D is upregulated in neurons while glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1-positive cells are upregulated in Mecp2T158A/y mice. Anti-SEMA4D treatment ameliorates Rett syndrome-specific symptoms and improves behavioural functions in both pre-symptomatic and symptomatic cohorts of hemizygous Mecp2T158A/y male mice. Anti-SEMA4D also reduces astrocyte and microglia activation in vivo. In vitro experiments demonstrate an abnormal cytoskeletal structure in mutant astrocytes in the presence of SEMA4D, while anti-SEMA4D antibody treatment blocks SEMA4D–Plexin B1 signaling and mitigates these abnormalities. These results suggest that anti-SEMA4D immunotherapy may be an effective treatment option to alleviate symptoms and improve cognitive and motor function in Rett syndrome.

SEMA4D Knockdown Attenuates β-Catenin-Dependent Tumor Progression in Colorectal Cancer

Semaphorin 4D (SEMA4D), a protein originally demonstrated to regulate the immune system and axonal growth cone collapse in the developing central nervous system, is overexpressed in various human malignancies, including colorectal cancer (CRC). This investigation was undertaken to examine the effects of SEMA4D silencing on the biological properties of the CRC cell line. SW48 cells were transfected with a siRNA-targeting SEMA4D. The mRNA expression of underlying pro- and antiapoptotic proteins including Bax, Bcl-2, P53, and caspase-3, cancer stem cell (CSC) markers, epithelial-mesenchymal transition (EMT) markers, MMP-2, and MMP-9 was examined using qRT-PCR. Further, the protein expression of E-cadherin and β-catenin was confirmed by Western blot. SW48 cell migration and MMP activity were detected using scratch and zymography analysis, respectively. Finally, the apoptosis rate was assessed via the flowcytometry test. SEMA4D knock-down was associated with a considerable suppression of in vitro cell viability, EMT-related genes, CSC markers, β-catenin signaling pathway, sphere-forming, cell migration, and MMP-2 activity as well as induction of apoptosis. This study identifies the inhibitory effects of SEMA4D gene silencing on tumor progression. Thereby, this might conclude a possible alternative to cancer therapy by targeting several prominent pathways involved in cancer through SEMA4D suppression.

Inhibition of Semaphorin 4D/Plexin-B1 Signal Inhibits the Subchondral Bone Loss in Early-Stage Osteoarthritis of Temporomandibular Joint

Subchondral bone loss is an important pathological feature of early-stage temporomandibular joint (TMJ) osteoarthritis (OA). Previous studies focused mainly on the bone resorption by osteoclasts in early-stage OA, but the bone formation feature has not drawn enough attention. Sema4D/Plexin-B1 is a pair of molecules expressed by osteoclast/osteoblast, which is capable of inhibiting bone formation by osteoblasts. The present study found that subchondral bone loss in early-stage TMJ OA was accompanied by up-regulated expression of Sema4D in cartilage and subchondral bone and Plexin-B1 in subchondral bone. Reducing Sema4D level could inhibit the subchondral bone loss and cartilage degeneration of early-stage TMJ OA. In vitro, results revealed that Sema4D could reduce the expression of osteocalcin (OCN) and alkaline phosphatase (ALP), and increase the migrating capability of Plexin-B1-positive osteoblasts. Our results revealed that elevated Sema4D expression in early-stage TMJ OA might decrease the bone formation activity of osteoblasts in the subchondral bone by binding to Plexin-B1 expressed by osteoblasts. Inhibiting Sema4D/Plexin-B1 signaling in the early-stage OA holds promise as a strategy for new therapeutic approaches to osteoarthritis.