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 Huntington's 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.

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.

CXCL13 antibody for the treatment of autoimmune disorders.

BACKGROUND: Homeostatic 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. RESULTS: We 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)). CONCLUSIONS: We developed a novel therapeutic antibody targeting CXCL13-mediated signaling pathway for the treatment of autoimmune disorders.

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.

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.

Malignant profile detected by CT angiographic information predicts poor prognosis despite thrombolysis within three hours from symptom onset.

OBJECTIVE: A malignant profile of early brain ischemia has been demonstrated in the Diffusion and Perfusion Imaging Evaluation for Understanding Stroke Evolution (DEFUSE) trial. Patients with a malignant profile had a low chance for an independent functional outcome despite thrombolysis within 3-6 h. We sought to determine whether CT angiography (CTA) could identify a malignant imaging profile within 3 h from symptom onset. METHODS: We studied consecutive patients (04/02-09/07) with anterior circulation stroke who received CTA before intravenous thrombolysis within 3 h. We assessed the Alberta Stroke Program Early CT Score (ASPECTS) on CTA source images (CTASI). Intracranial thrombus burden on CTA was assessed with a novel 10-point clot burden score (CBS). We analyzed percentages independent (modified Rankin Scale score < or =2) and fatal outcome at 3 months and parenchymal hematoma rates across categorized combined CTASI-ASPECTS + CBS score groups where 20 is best and 0 is worst. RESULTS: We identified 114 patients (median age 73 years [interquartile range 61-80], onset-to-tPA time 129 min [95-152]). Among 24 patients (21%) with extensive hypoattenuation on CTASI and extensive thrombus burden (combined score < or =10), only 4% (1/24) were functionally independent whereas mortality was 50% (12/24). In contrast, 57% (51/90) of patients with less affected scores (combined score 11-20) were functionally independent and mortality was 10% (9/90; p < 0.001). Parenchymal hematoma rates were 30% (7/23) vs. 8% (7/88), respectively (p = 0.008). CONCLUSION: CTA identifies a large hyperacute stroke population with high mortality and low likelihood for independent functional outcome despite early thrombolysis.