Semaphorin 4D is upregulated in neurons of diseased brains and triggers astrocyte reactivity.

BACKGROUND: The close interaction and interdependence of astrocytes and neurons allows for the possibility that astrocyte dysfunction contributes to and amplifies neurodegenerative pathology. Molecular pathways that trigger reactive astrocytes may represent important targets to preserve normal homeostatic maintenance and modify disease progression. METHODS: Semaphorin 4D (SEMA4D) expression in the context of disease-associated neuropathology was assessed in postmortem brain sections of patients with Huntington's (HD) and Alzheimer's disease (AD), as well as in mouse models of HD (zQ175) and AD (CVN; APPSwDI/NOS2-/-) by immunohistochemistry. Effects of SEMA4D antibody blockade were assessed in purified astrocyte cultures and in the CVN mouse AD model. CVN mice were treated weekly from 26 to 38 weeks of age; thereafter mice underwent cognitive assessment and brains were collected for histopathology. RESULTS: We report here that SEMA4D is upregulated in neurons during progression of neurodegenerative diseases and is a trigger of reactive astrocytes. Evidence of reactive astrocytes in close proximity to neurons expressing SEMA4D is detected in brain sections of patients and mouse models of HD and AD. We further report that SEMA4D-blockade prevents characteristic loss of GABAergic synapses and restores spatial memory and learning in CVN mice, a disease model that appears to reproduce many features of AD-like pathology including neuroinflammation. In vitro mechanistic studies demonstrate that astrocytes express cognate receptors for SEMA4D and that ligand binding triggers morphological variations, and changes in expression of key membrane receptors and enzymes characteristic of reactive astrocytes. These changes include reductions in EAAT-2 glutamate transporter and glutamine synthetase, key enzymes in neurotransmitter recycling, as well as reduced GLUT-1 glucose and MCT-4 lactate transporters, that allow astrocytes to couple energy metabolism with synaptic activity. Antibody blockade of SEMA4D prevented these changes and reversed functional deficits in glucose uptake. CONCLUSIONS: Collectively, these results suggest that SEMA4D blockade may ameliorate disease pathology by preserving normal astrocyte function and reducing the negative consequences of reactive astrogliosis.

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.

Effects of diet and exercise on adipocytokine levels in patients with moderate to severe chronic kidney disease.

BACKGROUND AND AIMS: Obesity is a pro-inflammatory risk factor for progression of CKD and cardiovascular disease. We hypothesized that implementation of caloric restriction and endurance exercise would improve adipocytokine profiles in patients with moderate to severe CKD. METHODS AND RESULTS: We enrolled patients with moderate to severe CKD through a multi-center pilot randomized trial of diet and exercise in a 4-arm design (dietary restriction of 10%-15% reduction in caloric intake, exercise three times/week, combined diet and exercise, and control) (NCT01150851). Adipocytokines (adiponectin and leptin) were measured at the beginning and end of the study period as secondary outcomes. Treatment effect was analyzed in a multivariable model adjusted for baseline outcome values, age, gender, site and diabetes. A total of 122 participants were consented, 111 were randomized (42% female, 25% diabetic, and 91% hypertensive), 104 started intervention and 92 completed the study (Figure 1). Plasma adiponectin levels increased significantly in response to diet by 23% (95% CI: 0.2%, 49.8%, p = 0.048) among participants randomized to the caloric restriction and usual activity arm but not to exercise, whereas circulating leptin did not change by either treatment. CONCLUSION: Our data suggest that dietary caloric restriction increases plasma adiponectin levels in stage 3-4 CKD patients, with limited effect on leptin levels. These findings suggest the potential for improving the metabolic milieu of CKD with moderate calorie restriction.

The effects of 16-weeks of prebiotic supplementation and aerobic exercise training on inflammatory markers, oxidative stress, uremic toxins, and the microbiota in pre-dialysis kidney patients: a randomized controlled trial-protocol paper.

BACKGROUND: Chronic kidney disease (CKD) is characterized by dysbiosis, elevated levels of uremic toxins, systemic inflammation, and increased markers of oxidative stress. These factors lead to an increased risk of cardiovascular disease (CVD) which is common among CKD patients. Supplementation with high amylose maize resistant starch type 2 (RS-2) can change the composition of the gut microbiota, and reduce markers of inflammation and oxidative stress in patients with end-stage renal disease. However, the impact of RS-2 supplementation has not been extensively studied in CKD patients not on dialysis. Aerobic exercise training lowers certain markers of inflammation in CKD patients. Whether combining aerobic training along with RS-2 supplementation has an additive effect on the aforementioned biomarkers in predialysis CKD patients has not been previously investigated. METHODS: The study is being conducted as a 16-week, double-blind, placebo controlled, parallel arm, randomized controlled trial. Sixty stage 3-4 CKD patients (ages of 30-75 years) are being randomized to one of four groups: RS-2 & usual care, RS-2 & aerobic exercise, placebo (cornstarch) & usual care and placebo & exercise. Patients attend four testing sessions: Two baseline (BL) sessions with follow up visits 8 (wk8) and 16 weeks (wk16) later. Fasting blood samples, resting brachial and central blood pressures, and arterial stiffness are collected at BL, wk8 and wk16. A stool sample is collected for analysis of microbial composition and peak oxygen uptake is assessed at BL and wk16. Blood samples will be assayed for p-cresyl sulphate and indoxyl sulphate, c-reactive protein, tumor necrosis factor α, interleukin 6, interleukin 10, monocyte chemoattractant protein 1, malondialdehyde, 8-isoprostanes F2a, endothelin-1 and nitrate/nitrite. Following BL, subjects are randomized to their group. Individuals randomized to conditions involving exercise will attend three supervised moderate intensity (55-65% peak oxygen uptake) aerobic training sessions (treadmills, bikes or elliptical machine) per week for 16 weeks. DISCUSSION: This study has the potential to yield information about the effect of RS-2 supplementation on key biomarkers believed to impact upon the development of CVD in patients with CKD. We are examining whether there is an additive effect of exercise training and RS-2 supplementation on these key variables. TRIAL REGISTRATION: Clinicaltrials.gov Trial registration# NCT03689569 . 9/28/2018, retrospectively registered.

SEMA4D compromises blood-brain barrier, activates microglia, and inhibits remyelination in neurodegenerative disease.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by immune cell infiltration of CNS, blood-brain barrier (BBB) breakdown, localized myelin destruction, and progressive neuronal degeneration. There exists a significant need to identify novel therapeutic targets and strategies that effectively and safely disrupt and even reverse disease pathophysiology. Signaling cascades initiated by semaphorin 4D (SEMA4D) induce glial activation, neuronal process collapse, inhibit migration and differentiation of oligodendrocyte precursor cells (OPCs), and disrupt endothelial tight junctions forming the BBB. To target SEMA4D, we generated a monoclonal antibody that recognizes mouse, rat, monkey and human SEMA4D with high affinity and blocks interaction between SEMA4D and its cognate receptors. In vitro, anti-SEMA4D reverses the inhibitory effects of recombinant SEMA4D on OPC survival and differentiation. In vivo, anti-SEMA4D significantly attenuates experimental autoimmune encephalomyelitis in multiple rodent models by preserving BBB integrity and axonal myelination and can be shown to promote migration of OPC to the site of lesions and improve myelin status following chemically-induced demyelination. Our study underscores SEMA4D as a key factor in CNS disease and supports the further development of antibody-based inhibition of SEMA4D as a novel therapeutic strategy for MS and other neurologic diseases with evidence of demyelination and/or compromise to the neurovascular unit.

Use of poxvirus display to select antibodies specific for complex membrane antigens.

Antibody discovery against complex antigens is limited by the availability of a reproducible pure source of concentrated properly folded antigen. We have developed a technology to enable direct incorporation of membrane proteins such as GPCRs and into the membrane of poxvirus. The protein of interest is correctly folded and expressed in the cell-derived viral membrane and does not require any detergents or refolding before downstream use. The poxvirus is selective in which proteins are incorporated into the viral membrane, making the antigen poxvirus an antigenically cleaner target for in vitro panning. Antigen-expressing virus can be readily purified at scale and used for antibody selection using any in vitro display platform.

Pepinemab antibody blockade of SEMA4D in early Huntington's disease: a randomized, placebo-controlled, phase 2 trial.

SIGNAL is a multicenter, randomized, double-blind, placebo-controlled phase 2 study (no. NCT02481674) established to evaluate pepinemab, a semaphorin 4D (SEMA4D)-blocking antibody, for treatment of Huntington's disease (HD). The trial enrolled a total of 265 HD gene expansion carriers with either early manifest (EM, n = 179) or late prodromal (LP, n = 86) HD, randomized (1:1) to receive 18 monthly infusions of pepinemab (n = 91 EM, 41 LP) or placebo (n = 88 EM, 45 LP). Pepinemab was generally well tolerated, with a relatively low frequency of serious treatment-emergent adverse events of 5% with pepinemab compared to 9% with placebo, including both EM and LP participants. Coprimary efficacy outcome measures consisted of assessments within the EM cohort of (1) a two-item HD cognitive assessment family comprising one-touch stockings of Cambridge (OTS) and paced tapping (PTAP) and (2) clinical global impression of change (CGIC). The differences between pepinemab and placebo in mean change (95% confidence interval) from baseline at month 17 for OTS were -1.98 (-4.00, 0.05) (one-sided P = 0.028), and for PTAP 1.43 (-0.37, 3.23) (one-sided P = 0.06). Similarly, because a significant treatment effect was not observed for CGIC, the coprimary endpoint, the study did not meet its prespecified primary outcomes. Nevertheless, a number of other positive outcomes and post hoc subgroup analyses-including additional cognitive measures and volumetric magnetic resonance imaging and fluorodeoxyglucose-positron-emission tomography imaging assessments-provide rationale and direction for the design of a phase 3 study and encourage the continued development of pepinemab in patients diagnosed with EM HD.

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T-cell Engagers Generates Antitumor Immunity While Avoiding Anergy.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

A Phase Ib/II Study of Pepinemab in Combination with Avelumab in Advanced Non-Small Cell Lung Cancer.

PURPOSE: The CLASSICAL-Lung clinical trial tested the combination of pepinemab, an IgG4 humanized mAb targeting semaphorin 4D, with the PD-L1 inhibitor avelumab to assess the effects of coupling increased T-cell infiltration and reversal of immune suppression via pepinemab with sustained T-cell activation via checkpoint inhibition. PATIENTS AND METHODS: This phase Ib/II, single-arm study was designed to evaluate the safety, tolerability, and efficacy of pepinemab in combination with avelumab in 62 patients with advanced non-small cell lung cancer (NSCLC), including immunotherapy-naïve (ION) patients and patients whose tumors progressed following anti-PD-1/L1 monotherapy (IOF). The main objectives were to evaluate safety/tolerability, establish a recommended phase 2 dose (RP2D), obtain a preliminary evaluation of antitumor activity, and investigate candidate biomarker activity. RESULTS: The combination was well tolerated with no major safety signals identified. Pepinemab, 10 mg/kg with avelumab, 10 mg/kg, every 2 weeks, was selected as the RP2D. Among 21 evaluable ION patients, 5 patients experienced partial responses (PR), 4 patients evidenced clinical benefit ≥1 year, and the disease control rate (DCR) was 81%. Notably, overall response rate with the combination therapy was higher than previously reported for single-agent avelumab in the PD-L1-negative/low population. Among 29 evaluable IOF patients, the combination resulted in a DCR of 59%, including 2 PR and 7 patients with durable clinical benefit of ≥23 weeks. Biomarker analysis of biopsies demonstrated increased CD8 T-cell density correlating with RECIST response criteria. CONCLUSIONS: The combination of pepinemab with avelumab was well tolerated in NSCLC and showed signs of antitumor activity in immunotherapy-resistant and PD-L1-negative/low tumors.

Semaphorin4D Inhibition Improves Response to Immune-Checkpoint Blockade via Attenuation of MDSC Recruitment and Function.

Tumor infiltration by immunosuppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs), causes resistance to immunotherapy. Semaphorin4D, originally characterized for its axonal guidance properties, also contributes to endothelial cell migration and survival and modulates global immune cytokine profiles and myeloid cell polarization within the tumor microenvironment. Here, we show how a therapeutic murine Sema4D mAb improves responses to immune-checkpoint blockade (ICB) in two murine carcinoma models. Treatment of tumor-bearing mice with Sema4D mAb abrogated Ly6Ghi PMN-MDSC recruitment through reducing MAPK-dependent chemokine production by tumor cells in Murine oral cancer-1 (MOC1) tumors. PMN-MDSC suppressive capacity was reduced through inhibition of Sema4D-driven arginase expression. These changes led to enhanced tumor infiltration by CD8+ TIL and activation of tumor-draining lymph node T lymphocytes in response to tumor antigen. Sema4D mAb in combination with either CTLA-4 or PD-1 blockade enhanced rejection of tumors or tumor growth delay, resulting in prolonged survival with either treatment. This function of Sema4D mAb provides a rationale for its evaluation in combination with ICB to treat tumors with immunosuppressive myeloid infiltration.

C35 (C17orf37) is a novel tumor biomarker abundantly expressed in breast cancer.

Identification of shared tumor-specific targets is useful in developing broadly applicable therapies. In a study designed to identify genes up-regulated in breast cancer, a cDNA clone corresponding to a novel gene C35 (C17orf37) was selected by representational difference analysis of tumor and normal human mammary cell lines. Abundant expression of C35 transcript in tumors was confirmed by Northern blot and real-time PCR. The C35 gene is located on chromosome 17q12, 505 nucleotides from the 3' end of the ERBB2 oncogene, the antigenic target for trastuzumab (Herceptin) therapy. The chromosomal arrangement of the genes encoding C35 and ERBB2 is tail to tail. An open reading frame encodes a 12-kDa protein of unknown function. Immunohistochemical analysis detected robust and frequent expression of C35 protein, including 32% of grade 1 and 66% of grades 2 and 3 infiltrating ductal carcinomas of the breast (in contrast to 20% overexpressing HER-2/neu), 38% of infiltrating lobular carcinoma (typically HER-2/neu negative), as well as tumors arising in other tissues. C35 was not detected in 38 different normal human tissues, except Leydig cells in the testes and trace levels in a small percentage of normal breast tissue samples. The distinct and favorable expression profile of C35 spanning early through late stages of disease, including high frequency of overexpression in various breast carcinoma, abundant expression in distant metastases, and either absence or low level expression in normal human tissues, warrants further investigation of the relevance of C35 as a biomarker and/or a target for development of broadly applicable cancer-specific therapies.

Immunomodulation of the tumor microenvironment by neutralization of Semaphorin 4D.

Semaphorin 4D is highly expressed at the invasive tumor margin and acts as a guidance molecule, restricting movement of tumoricidal immune cells into the tumor microenvironment. We recently showed that antibody neutralization of SEMA4D augmented activated monocyte and anticancer T-cell tumor penetration and that anti-SEMA4D antibody potentiated other immunomodulatory therapies in murine tumor models.

Antibody Blockade of Semaphorin 4D Promotes Immune Infiltration into Tumor and Enhances Response to Other Immunomodulatory Therapies.

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 (PLXNB1) are broadly expressed in murine and human tumors, and their expression has been shown to correlate with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, SEMA4D-PLXNB1 interactions have been reported to affect vascular stabilization and transactivation of ERBB2, but effects on immune-cell trafficking in the tumor microenvironment (TME) have not been investigated. We describe a novel immunomodulatory function of SEMA4D, whereby strong expression of SEMA4D at the invasive margins of actively growing tumors influences the infiltration and distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient of expression, enhances recruitment of activated monocytes and lymphocytes into the tumor, and shifts the balance of cells and cytokines toward a proinflammatory and antitumor milieu within the TME. This orchestrated change in the tumor architecture was associated with durable tumor rejection in murine Colon26 and ERBB2(+) mammary carcinoma models. The immunomodulatory activity of anti-SEMA4D antibody can be enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival. Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the TME and inhibit tumor progression.