66 - Proof-of-concept evaluation of intravenous anti-CD47 antibodies for augmentation of hematoma clearance and mitigation of acute neuropathology in a rat model of penetrating traumatic brain injury

Abstract Text: Background. Therapeutics for traumatic brain injury (TBI)-induced intracerebral hematoma represent a critical gap for battlefield point-of-injury care as there are currently no effective treatment options. CD47 is an anti-phagocytic cell surface protein that inhibits hematoma clearance. Animal studies have shown that inhibition of CD47 can reduce lesion volume, brain edema, neuronal loss, and improved neurological outcomes in preclinical studies of ischemic brain injury. This project was designed to test the ability of anti-CD47 antibodies to promote hematoma clearance in a model of severe TBI with intracerebral hemorrhage. The objectives of this proof-of-concept study was to evaluate the pharmacokinetic and pharmacodynamics (PK/PD) profile of intravenously (i.v.) administrated anti-CD47 antibody following severe TBI as well as assess hematoma clearance, and neuroprotective effects. We hypothesized that inhibition of TBI-induced upregulation of CD47 mediated anti-phagocytic mechanisms will be well tolerated, augment hematoma clearance and mitigate acute neuropathology.

Methods. Two animal cohorts (72 hrs and 7 days post injury) were evaluated. Sprague-Dawley rats underwent right jugular vein cannulation and received a frontal, unilateral penetrating ballistic-like brain injury (PBBI). Following PBBI, anti-CD47 antibody conjugated to FITC or equal volume vehicle was delivered in a single i.v. dose at control, 0.1, 0.2, or 0.5 mg antibodies/kg of body weight. Blood was collected for serum chemistry panels, complete blood count, and antibody quantitation at 30 min, 6 hrs, 24 hrs, 48 hrs, 72 hrs, and 7 days post-administration. Brain tissue was also collected for antibody quantitation at 72 hrs post-injury. Anti-CD47 antibody concentration in plasma and brain lysates (ipsilateral/contralateral) was quantitated using an optimized fluorescence-based spectrophotometric assay. An extensive diagnostic necropsy was also performed 72 hrs post-injury to assess effects of treatment on heart, lung, liver, spleen, kidney, and bone marrow. To determine efficacy on augmenting hematoma clearance and mitigating TBI-induced neuropathology at 72 hrs, neurological deficits were evaluated daily using a modified Bedersons neurological assessment. Brain tissue at 72 hrs post-injury and treatment was also collected for cerebral edema measurement, histological preparation, and hemoglobin assay. Cerebral edema was measured using the wet-dry method. Histology was performed for determination of neuroinflammation (GFAP, and IBA1). Hemoglobin content was quantified using an enzyme-linked immunoassay for evaluation of hematoma clearance.

Results. The PK profile of anti-CD47 antibody elicited that antibody concentration decayed over 7 days post-administration. Anti-CD47 antibody levels achieved following a single dose of 0.1 mg/kg was still present in the blood (0.568±0.098 ug/ml) at 72 hrs post-administration. Blood tests and necropsy analysis indicated no severe adverse events following treatment. Cerebral hemoglobin levels were significantly increased after injury, however, anti-CD47 antibody administration at 0.1 mg/kg resulted in a trending reduction in cerebral hemoglobin levels at 72 hrs post-administration, indicating augmentation of intracerebral hematoma clearance. The administration of anti-CD47 antibody did not exacerbate or improve neurological deficits observed following PBBI. PBBI elicited an increase in cerebral edema which was not improved by anti-CD47 administration. Immunohistochemistry assessment of GFAP and IBA1 demonstrated a significant reduction of GFAP levels in the lesion core at 0.1 mg/kg (** P< 0.01) and peri-lesional area at both 0.1 mg/kg (**P < 0.01) and 0.2 mg/kg doses (*P < 0.05) as compared to control. IBA1 levels were not affected by treatment.

Conclusions. These results provide evidence that peripherally administrated anti-CD47 antibody following penetrating TBI did not cause severe adverse events and were well tolerated. While no improvement of acute neurological deficits was observed, analysis of neuropathology showed both neuroprotective effects and hematoma clearance following anti-CD47 antibody administration at lower dose (0.1 mg/kg), which may suggest a potential benefit to treatment following penetrating TBI.

Support. This work was supported by Combat Casualty Care Research Program (CO210009).

Keywords: intracerebral hematoma clearance, inhibition of CD47, pharmacokinetics and pharmacodynamics, intravenous anti-CD47 antibodies, neuroprotection