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.

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.

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.

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.

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.

Frequency and clinical course of stroke and transient ischemic attack patients with intracranial nonocclusive thrombus on computed tomographic angiography.

BACKGROUND AND PURPOSE: We sought to determine the frequency and clinical course of patients with acute ischemic stroke or transient ischemic attack (TIA) who had intracranial nonocclusive thrombus (iNOT) on CT angiography (CTA). METHODS: We retrospectively (June 2002-March 2007) reviewed consecutive patients with acute ischemic stroke or TIA who had CTA performed acutely for diagnostic work-up. A neuroradiologist reviewed all cases with potential iNOT. Criteria to diagnose iNOT rather than occlusive thrombus or atherosclerotic stenosis were: (1) residual lumen present and eccentric; (2) nontapering thrombus; (3) smooth and well-defined thrombus margins; and (4) absence of vessel wall calcification. We defined functional independence at discharge as modified Rankin scale score

Normal magnetic resonance perfusion-weighted imaging in lacunar infarcts predicts a low risk of early deterioration.

BACKGROUND: Current clinical tools to identify lacunar infarct patients at risk of deterioration are inadequate, and imaging techniques to predict fluctuation and deterioration would be of value. We sought to determine the occurrence of MRI perfusion-weighted imaging (PWI) abnormalities in lacunes, and whether they help predict clinical and radiological outcome. METHODS: Patients with lacunar stroke or TIA were selected from a prospective MR imaging study. MRI was performed within 24 h of the event and follow-up imaging completed at 30 or 90 days. Baseline perfusion maps were qualitatively assessed and infarct volumes measured. Early clinical deterioration (NIHSS worsening of > or = 3 points within 72 h of event) and 90-day modified Rankin Scale score (mRS) were recorded. RESULTS: Twenty-two patients were included. Fifteen (68.2%) had abnormal PWI at the site of the diffusion-weighted imaging lesion. Patients with abnormal PWI were more likely to have stroke than TIA as their index event (RR 2.2, 95% CI 0.9-5.2, p = 0.02). Early clinical deterioration occurred in 4 patients (18.2%), all of whom had abnormal PWI. PWI lesions were not associated with a higher 90-day NIHSS or mRS score, nor did they predict infarct volume growth. CONCLUSIONS: MR-PWI abnormalities are seen in two thirds of lacunar infarcts, and are associated with stroke rather than TIA. Normal PWI identifies patients at low risk of early clinical deterioration.

Does intravenous rtPA benefit patients in the absence of CT angiographically visible intracranial occlusion?

BACKGROUND: In patients with acute stroke receiving intravenous tissue plasminogen activator (tPA), we postulated that the presence of intracranial occlusion on CT angiography (CTA) modifies the benefit of thrombolysis. MATERIALS AND METHODS: Using a retrospective cohort design, we identified patients with acute ischemic stroke in our CTA database between May 2002 and August 2007. All the patients had a CTA within 12 h of onset, a premorbid modified Rankin scale (mRS) < or = 1, and a baseline National Institute of Health Stroke Scale score(NIHSS)f > or = 6. The primary outcome was early effectiveness of tPA defined as an NIHSS score of < or = 2 at 24 h or a 4-point NIHSS improvement at 24 h. Secondary outcome included mRS < or = 1 at 90 days. The relationship between intracranial occlusion on CTA and benefit of tPA was assessed using a test for interaction. RESULTS: A total of 287 patients met the criteria [occlusion present N =168; (98 with tPA; 70 without tPA) and occlusion absent N = 119; (52 with tPA; 67 without tPA)]. Those with intracranial occlusion were likely to have more severe strokes (NIHSS > or = 15; P < 0.001) and abnormal brain imaging (ASPECTS < or =7; P < 0.001). For outcome of 4-point NIHSS score improvement at 24 h, benefit from tPA was observed only among patients with a visible occlusion (absolute difference in favor of tPA: 20.4% vs. 0.7%; P = 0.06). CONCLUSION: In patients with acute ischemic stroke, thrombolysis produced a better early clinical response among patients with intracranial occlusion, which needs to be confirmed in stroke thrombolysis trials.

Extent of hypoattenuation on CT angiography source images predicts functional outcome in patients with basilar artery occlusion.

BACKGROUND AND PURPOSE: Quantification of early ischemic changes (EIC) may predict functional outcome in patients with basilar artery occlusion (BAO). We tested the validity of a novel CT score, the posterior circulation Acute Stroke Prognosis Early CT score (pc-ASPECTS). METHODS: Pc-ASPECTS allots the posterior circulation 10 points. Two points each are subtracted for EIC in midbrain or pons and 1 point each for EIC in left or right thalamus, cerebellum or PCA-territory, respectively. We studied 2 different populations: (1) patients with suspected vertebrobasilar ischemia and (2) patients with BAO. We applied pc-ASPECTS to noncontrast CT (NCCT), CT angiography source images (CTASI), and follow-up image by 3-reader consensus. We calculated sensitivity for ischemic changes and analyzed the predictivity of pc-ASPECTS for independent (modified Rankin Scale [mRS] score /=8 but only 4% (1/23) with a score <8 had favorable functional outcome (RR 12.1; 95% CI, 1.7 to 84.9). This difference was consistent in 21 patients with angiographic recanalization (RR 7.7; 95% CI, 1.1 to 52.1). CONCLUSIONS: The CTASI pc-ASPECTS score may identify BAO patients unlikely to have a favorable outcome despite recanalization.

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. CLINICALTRIALSGOV 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%).

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

BACKGROUND: 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. METHODS: The assay detects VX15/2503, a human IgG4 specific for SEMA4D, with an IgG4 -specific monoclonal antibody. RESULTS: Data generated allowed assessment of two related SEMA4D-specific pharmacodynamic (PD) markers: (1) The measurement of cellular SEMA4D (cSEMA4D) saturation by VX15/2503, and (2) the cell membrane expression levels of cSEMA4D. CONCLUSIONS: This assay specifically and reproducibly measured cSEMA4D saturation and expression levels. Evaluation of the SEMA4D-specific PD markers were critical in determining the clinical saturation threshold of cSEMA4D by VX15/2503.

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.

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.

Cognitive therapy: a training model for advanced practice nurses.

1. Mental health needs exist in medically underserved areas and can be addressed in nurse-managed, community-based health centers. 2. Cognitive therapy techniques can be used in community-based health centers to intervene and alleviate patients' distress and improve their adherence to treatment. 3. A training program in cognitive therapy can help advanced practice nurses and other health care providers implement the techniques needed to address many behavioral and mental health problems.

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.

Influence of adiposity and physical activity on arterial stiffness in healthy children: the lifestyle of our kids study.

Childhood obesity is increasingly prevalent in the community and is related to adverse cardiovascular outcomes during adulthood. In this study of healthy children, we evaluated the influence of adiposity and physical activity on carotid-femoral pulse wave velocity (PWV), an index of arterial stiffness and a marker of cardiovascular risk in adults. In 573 community-based children (mean age: 10.1+/-0.3 years; 51% boys), we measured body mass index and waist circumference. Percentage body fat was quantitated by dual-energy x-ray absorptiometry. Cardiorespiratory fitness (CRF) and physical activity levels were assessed using a 20-m shuttle run and 7-day pedometer count, respectively. PWV was estimated by applanation tonometry. In univariate analysis, PWV was positively correlated with body mass index (r=0.34), waist circumference (r=0.32), and percentage body fat (r=0.32; P<0.001 for all) and negatively correlated with CRF (r=-0.23; P<0.001) and pedometer count (r=-0.08; P=0.046). In separate multivariable linear regression models, body mass index, waist circumference, and percentage of body fat were independently and positively associated with PWV (P<0.01 for all) after adjusting for age, sex, systolic blood pressure, mean arterial pressure, heart rate, and CRF (P<0.01 for all). The influence of CRF on PWV was attenuated after adjusting for adiposity. In conclusion, increased body mass and adiposity and decreased CRF are associated with arterial stiffening in healthy prepubescent children.

Intracranial thrombus extent predicts clinical outcome, final infarct size and hemorrhagic transformation in ischemic stroke: the clot burden score.

BACKGROUND: In ischemic stroke, functional outcomes vary depending on site of intracranial occlusion. We tested the prognostic value of a semiquantitative computed tomography angiography-based clot burden score. METHODS: Clot burden score allots major anterior circulation arteries 10 points for presence of contrast opacification on computed tomography angiography. Two points each are subtracted for thrombus preventing contrast opacification in the proximal M1, distal M1 or supraclinoid internal carotid artery and one point each for M2 branches, A1 and infraclinoid internal carotid artery. We retrospectively studied patients with disabling neurological deficits (National Institute of Health Stroke Scale score >or=5) and computed tomography angiography within 24-hours from symptom onset. We analyzed percentages independent functional outcome (modified Rankin Scale score