Christine Reilly

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.

CXCL13 antibody for the treatment of autoimmune disorders

BackgroundHomeostatic B Cell-Attracting chemokine 1 (BCA-1) otherwise known as CXCL13 is constitutively expressed in secondary lymphoid organs by follicular dendritic cells (FDC) and macrophages. It is the only known ligand for the CXCR5 receptor, which is expressed on mature B cells, follicular helper T cells (Tfh), Th17 cells and regulatory T (Treg) cells. Aberrant expression of CXCL13 within ectopic germinal centers has been linked to the development of autoimmune disorders (e.g. Rheumatoid Arthritis, Multiple Sclerosis, Systemic Lupus Erythematosis). We, therefore, hypothesized that antibody-mediated disruption of the CXCL13 signaling pathway would interfere with the formation of ectopic lymphoid follicles in the target organs and inhibit autoimmune disease progression. This work describes pre-clinical development of human anti-CXCL13 antibody MAb 5261 and includes therapeutic efficacy data of its mouse counterpart in murine models of autoimmunity.ResultsWe developed a human IgG1 monoclonal antibody, MAb 5261 that specifically binds to human, rodent and primate CXCL13 with an affinity of approximately 5 nM and is capable of neutralizing the activity of CXCL13 from these various species in in vitro functional assays. For in vivo studies we have engineered a chimeric antibody to contain the same human heavy and light chain variable genes along with mouse constant regions. Treatment with this antibody led to a reduction in the number of germinal centers in mice immunized with 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Keyhole Limpet Hemocyanin (NP-KLH) and, in adoptive transfer studies, interfered with the trafficking of B cells to the B cell areas of mouse spleen. Furthermore, this mouse anti-CXCL13 antibody demonstrated efficacy in a mouse model of Rheumatoid arthritis (Collagen-Induced Arthritis (CIA)) and Th17-mediated murine model of Multiple Sclerosis (passively-induced Experimental Autoimmune Encephalomyelitis (EAE)).ConclusionsWe developed a novel therapeutic antibody targeting CXCL13-mediated signaling pathway for the treatment of autoimmune disorders.

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

Evans and colleagues describe a novel immunomodulatory function of semaphorin 4D (SEMA4D) and show that blocking SEMA4D enhances immune infiltration into tumor and increases antitumor activity in synergy with 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. Cancer Immunol Res; 3(6); 689–701. ©2015 AACR.

Safety, Pharmacokinetics and Pharmacodynamics of a Humanized anti-Semaphorin 4D Antibody, in a First-In-Human Study of Patients with Advanced Solid Tumors

Purpose: Study objectives included evaluating the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of VX15/2503 in advanced solid tumor patients. Experimental Design: Weekly i.v. doses were administered on a 28-day cycle. Safety, immunogenicity, PK, efficacy, T-cell membrane–associated SEMA4D (cSEMA4D) expression and saturation, soluble SEMA4D (sSEMA4D) serum levels, and serum biomarker levels were evaluated. Results: Forty-two patients were enrolled into seven sequential cohorts and an expansion cohort (20 mg/kg). VX15/2503 was well tolerated. Treatment-related adverse events were primarily grade 1 or 2 and included nausea (14.3%) and fatigue (11.9%); arthralgia, decreased appetite, infusion-related reaction, and pyrexia were each 7.3%. One pancreatic cancer patient (15 mg/kg) experienced a Grade 3 dose-limiting toxicity; elevated γ-glutamyl transferase. Complete cSEMA4D saturation was generally observed at serum antibody concentrations ≥0.3 μg/mL, resulting in decreased cSEMA4D expression. Soluble SEMA4D levels increased with dose and infusion number. Neutralizing anti-VX15/2503 antibodies led to treatment discontinuation for 1 patient. VX15/2503 Cmax and AUC generally increased with dose and dose number. One patient (20 mg/kg) experienced a partial response, 19 patients (45.2%) exhibited SD for ≥8 weeks, and 8 (19%) had SD for ≥16 weeks. Subjects with elevated B/T lymphocytes exhibited longer progression-free survival. Conclusions: VX15/2503 was well tolerated and produced expected PD effects. The correlation between immune cell levels at baseline and progression-free survival is consistent with an immune-mediated mechanism of action. Future investigations will be in combination with immunomodulatory agents. Clin Cancer Res; 22(4); 827–36. ©2015 AACR.

Nonclinical Safety Evaluation of VX15/2503, a Humanized IgG4 Anti-SEMA4D Antibody

The humanized IgG4 monoclonal antibody VX15/2503 bound with 1 to 5 nmol/L affinity to purified recombinant semaphorin 4D (SEMA4D; CD100) produced using murine, rat, cynomolgus macaque, and human sequences. The affinity for native SEMA4D expressed on macaque T lymphocytes was approximately 0.6 nmol/L. Tissues from rats and cynomolgus macaques demonstrated specific staining only with resident lymphocytes. Single-dose and one-month toxicology/PK studies used VX15/2503 dose levels of 0 to 100 mg/kg. No toxicity was observed with either species in these studies, thus the no observed adverse effect level (NOAEL) was 100 mg/kg. Cmax, exposure, and half-life values were similar for both rats and macaques. The NOAEL in a primate maximum feasible dose study was 200 mg/kg. Saturation of T-cell–associated SEMA4D occurred following administration of single doses of 0.1 mg/kg and above; five weekly injections of VX15/2503 at a dose level of 100 mg/kg produced saturation lasting for more than 120 and 130 days, respectively, for rats and primates. Macaques administered five weekly doses of VX15/2503 showed dose-dependent reductions of 2- to 3-fold in T-cell SEMA4D (cSEMA4D) expression levels compared with controls. Reduced cSEMA4D expression levels continued until serum antibody concentrations were 2 to 5 μg/mL, and thereafter normal cSEMA4D levels were restored. On the basis of these data, a phase I clinical study of the safety and tolerability of VX15/2503 was conducted, enrolling adult patients with advanced solid tumor diseases; a single-dose, dose escalation, phase I safety study was also initiated with subjects with multiple sclerosis. Mol Cancer Ther; 14(4); 964–72. ©2015 AACR.

Safety/tolerability of the anti-semaphorin 4D antibody VX15/2503 in a randomized phase 1 trial

Objective: To evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of VX15/2503 in a randomized, single-dose, dose-escalation, double-blind, placebo-controlled study enrolling adult patients with MS. Methods: Single IV doses of VX15/2503 or placebo were administered. Ten patients each were randomized (4:1 randomization ratio) into 5 ascending dose cohorts of 1, 3, 6, 10, or 20 mg/kg. Safety, immunogenicity, PK/PD, MRI, ECG, and lymphocyte subset levels were evaluated. A Dose Escalation Safety Committee (DESC) approved each dose escalation. Results: VX15/2503 was well tolerated, and all participants completed the study. Antibody treatment–related adverse events were primarily grade 1 or 2 and included urinary tract infection (12.5%) and muscle weakness, contusion, and insomnia (each 7.5%). No dose-limiting toxicities were observed, and no maximum tolerated dose was determined. One subject (20 mg/kg) experienced disease relapse 3 months before study entry and exhibited a grade 3 (nonserious) increase in brain lesions by day 29, possibly related to VX15/2503. Twenty-nine patients exhibited human anti-humanized antibody responses; 5 with titer ≥100. No anti-VX15/2503 antibody responses were fully neutralizing. VX15/2503 Cmax, area under the time-concentration curve, and mean half-life increased with dose level; at 20 mg/kg, the T1/2 was 20 days. Cellular SEMA4D saturation occurred at serum antibody concentrations ≤0.3 μg/mL, resulting in decreased cSEMA4D expression. At 20 mg/kg, cSEMA4D saturation persisted for ≥155 days. Total sSEMA4D levels increased with dose level and declined with antibody clearance. Conclusions: These results support the continued investigation of VX15/2503 in neurodegenerative diseases. ClinicalTrials.gov identifier: NCT01764737. Classification of evidence: This study provides Class III evidence that anti-semaphorin 4D antibody VX15/2503 at various doses was safe and well tolerated vs placebo, although an increase in treatment-emergent adverse events in the treatment group could not be excluded (risk difference −0.7%, 95% CI −28.0% to 32.7%).

Generation and preclinical characterization of an antibody specific for SEMA4D.

Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. Blocking the binding of SEMA4D to its receptors can result in physiologic changes that may have implications in cancer, autoimmune, and neurological disease. To study the effects of blocking SEMA4D, we generated, in SEMA4D-deficient mice, a panel of SEMA4D-specific hybridomas that react with murine, primate, and human SEMA4D. Utilizing the complementarity-determining regions from one of these hybridomas (mAb 67-2), we generated VX15/2503, a humanized IgG4 monoclonal antibody that is currently in clinical development for the potential treatment of various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntingtons disease. This work describes the generation and characterization of VX15/2503, including in vitro functional testing, epitope mapping, and an in vivo demonstration of efficacy in an animal model of rheumatoid arthritis.

Saturation monitoring of VX15/2503, a novel semaphorin 4D-specific antibody, in clinical trials

Receptor occupancy, or saturation, assays are often utilized in preclinical and clinical development programs to evaluate the binding of a biologic to a cellular target. These assays provide critical information regarding the dose of drug required to “saturate” the target as well as important pharmacodymamic (PD) data. A flow cytometric method was developed to measure the degree of Semaphorin 4D (SEMA4D; CD100) saturation by VX15/2303, an investigational monoclonal antibody specific for SEMA4D.

Antibody blockade of semaphorin 4D breaks down barriers to enhance tumoricidal immune infiltration and supports rational immunotherapy combinations

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates 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, we describe a novel immunomodulatory function of SEMA4D in regulating immune cell infiltration and anti-tumor activity. Activity is enhanced in preclinical studies when combined with other immunotherapies, including immune checkpoint blockade inhibition.

Abstract 3661: Breaking down the barrier restricting infiltration and differentiation of APC in the tumor microenvironment with a first-in-class antibody targeting Semaphorin4D, and rational combination therapies

Purpose: We expand mechanistic findings in preclinical studies to demonstrate that antibody blockade of Semaphorin 4D (SEMA4D, CD100) reduces expansion of MDSC and shifts the balance of myeloid cells within the TME to facilitate tumor rejection. Efficacy is further enhanced when combined with various immunotherapies. Design of Phase 1b/2 combination trials of VX15/2503, a humanized IgG4 antibody targeting SEMA4D, with immune checkpoint inhibition will be presented. Methods: Anti-SEMA4D antibodies were evaluated alone and in combination with other immunotherapies in various preclinical models. Anti-tumor activity and immune response was characterized by immunohistochemistry, flow cytometry, functional assays, and cytokine, chemokine and gene expression analysis. A Phase I trial for single agent VX15/2503 was completed, and several 1b/2 combination immunotherapy trials are planned. Results: SEMA4D restricts migration of monocytes and promotes expansion of suppressive myeloid cells in vitro. Strong expression of SEMA4D at the invasive margins of actively growing tumors in vivo restricts the infiltration and modulates polarization of leukocytes in the TME. Antibody blockade of SEMA4D facilitated recruitment of activated DCs and T lymphocytes in preclinical models. M-MDSCs were significantly reduced in tumor and blood following treatment. A significant shift towards increased Th1 cytokines (IFNγ, TNFα) and CTL-recruiting chemokine CXCL9, with concurrent reduction in Treg-, MDSC- and M2-macrophage promoting chemokines (CCL2, CXCL1, CXCL5) was observed. Accordingly, Teff:Treg ratio (3x, p Conclusions: SEMA4D blockade represents a novel mechanism to promote functional immune infiltration into the tumor and enhance immunotherapy. VX15/2503 treatment was well tolerated in a Phase I multiple ascending dose trial in patients with advanced refractory solid tumors. Phase 1b/2 trials of combination therapy with avelumab in NSCLC patients who are immunotherapy naive, and combinations with anti- anti-PD-1 and/or anti-CTLA-4 in melanoma and HNSCC patients who are refractory to PD1 inhibitors are planned in 2017. Citation Format: Elizabeth E. Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Maria Scrivens, Cathie Foster, Alan Howell, Stephen R. Comeau, Leslie Balch, Alyssa Knapp, John E. Leonard, Terrence L. Fisher, Siwen Hu-Lieskovan, Antoni Ribas, Ernest S. Smith, Maurice Zauderer. Breaking down the barrier restricting infiltration and differentiation of APC in the tumor microenvironment with a first-in-class antibody targeting Semaphorin4D, and rational combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3661. doi:10.1158/1538-7445.AM2017-3661