Administration of AAV-based gene therapies into the intra-cerebrospinal fl uid (CSF) compartments via routes such as lumbar puncture (LP) has been implemented as an alternative to intravenous dosing to target the CNS regions. This route enables lower doses, decreases systemic toxicity, and circumvents intravascular pre-existing anti-AAV antibodies. In this study, AAV9-GFP vectors were administered via LP to juvenile cynomolgus macaques with and without pre-existing serum antiAAV9 antibodies at a 5.0 x 1013 vector genomes per mL (vg/mL) dose and examined for 28 days. CNS and peripheral tissues were surveyed for vector genome, mRNA, and protein expression. Histopathology, clinical pathology, and humoral immune response to the viral capsid and transgene were also assessed. In addition, serum and CSF samples were analyzed to examine 276 proteomic markers curated to evaluate neural injury, organ damage, and inflammatory response. This study reveals no noticeable difference in AAV9-mediated gene transfer in the CNS tissues in the two groups; however, differences were observed for endpoints such as liver enzyme activities, histopathology, and levels of protein markers in the serum and CSF. These fi ndings provide a view into vector transduction effi ciency and safety following LP-delivered AAV9 to juvenile cynomolgus macaques with and without pre-existing antiAAV9 antibodies.

Supplementary Figures S1-S3.

Spinal muscular atrophy type 1 (SMA1) is a debilitating neurodegenerative disease resulting from survival motor neuron 1 gene (SMN1) deletion/mutation. Onasemnogene abeparvovec (formerly AVXS-101) is a gene therapy that restores SMN production via one-time systemic administration. The present study demonstrates widespread biodistribution of vector genomes and transgenes throughout the central nervous system (CNS) and peripheral organs, after intravenous administration of an AAV9-mediated gene therapy. Two symptomatic infants with SMA1 enrolled in phase III studies received onasemnogene abeparvovec. Both patients died of respiratory complications unrelated to onasemnogene abeparvovec. One patient had improved motor function and the other died shortly after administration before appreciable clinical benefit could be observed. In both patients, onasemnogene abeparvovec DNA and messenger RNA distribution were widespread among peripheral organs and in the CNS. The greatest concentration of vector genomes was detected in the liver, with an increase over that detected in CNS tissues of 300-1,000-fold. SMN protein, which was low in an untreated SMA1 control, was clearly detectable in motor neurons, brain, skeletal muscle and multiple peripheral organs in treated patients. These data support the fact that onasemnogene abeparvovec has effective distribution, transduction and expression throughout the CNS after intravenous administration and restores SMN expression in humans.

Abstract Purpose: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, whereas in a tumor Ang2 antagonizes Ang1's function and promotes the initiation of the angiogenic switch. Specifically targeting Ang2 is a promising anticancer strategy. Here we describe the development and characterization of a new class of biotherapeutics referred to as CovX-Bodies, which are created by chemical fusion of a peptide and a carrier antibody scaffold. Experimental Design: Various linker tethering sites on peptides were examined for their effect on CovX-Body in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nmol/L IC50s and significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in combination with standard of care agents. Tumor samples were analyzed for target engagement, via Ang2 protein level, CD31-positive tumor vasculature, and Tie2 expressing monocyte penetration. Results: Bivalent Ang2 CovX-Bodies selectively block the Ang2–Tie2 interaction (IC50 < 1 nmol/L) with dramatically improved pharmacokinetics (T½ > 100 hours). Using a staged Colo-205 xenograft model, significant tumor growth inhibition (TGI) was observed (40%–63%, P < 0.01). Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), whereas tumor microvessel density (P < 0.01) and intratumor proangiogenic Tie2+CD11b+ cells (P < 0.05) were significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang2 CovX-Bodies produced even greater efficacy (∼80% TGI, P < 0.01). Conclusion: CovX-Bodies provide an elegant solution to overcome the pharmacokinetic–pharmacodynamic problems of peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in combination with standard-of-care agents. Clin Cancer Res; 17(5); 1001–11. ©2011 AACR.

CVX-045 is produced by covalently attaching a thrombospondin 1 (TSP-1) mimetic comprising a peptidic sequence and a linker to the Fab binding site of a proprietary scaffold antibody. CVX-045 possesses the potency of the TSP-1-derived peptide, along with the advantageous pharmacokinetics of an antibody. Antitumor activity of CVX-045 was evaluated in human xenograft models alone and in combination with standard chemotherapies and targeted molecules. In A549 and A431 xenograft models, CVX-045 demonstrated significant (P < .05) antiangiogenic activity, reducing tumor microvessel density and increasing the levels of necrosis within treated tumors. In an HT-29 xenograft model, CVX-045 in combination with 5-fluorouracil significantly (P < .01) decreased tumor growth rate compared with vehicle, CVX-045, or 5-fluorouracil alone. Cotreatment of CVX-045 plus CPT-11 delayed progression of tumor growth from day 28 to 60. In contrast CVX-045 alone treatment did not delay the progression of tumor growth, and CPT-11 alone delayed progression of tumor growth to day 39. Cotreatment of CVX-045 with sunitinib extended the time to reach tumor load from day 26 to 40. In summary, CVX-045 exhibits significant antiangiogenic activity in several tumor models and enhances antitumor activity in combination with chemotherapy or targeted therapies. These data suggest future avenues for effective combination therapy in treating solid tumors. CVX-045 has recently completed a phase 1 trial in solid tumors where it has been well tolerated.

Abstract FGFR4 is the receptor for FGF19. Published data has shown FGF19 transgenic animals develop hepatocellular carcinoma (HCC), suggesting that the FGFR4:FGF19 axis may play a role in HCC. FGFR4 upregulation has been shown to be a negative prognostic indicator in multiple tumor types. CVX-63, an FGFR4-specific CovX-Body, was produced by fusing a chemically modified peptide to the specific Fab binding site of a specially designed antibody. CVX-63 was shown to inhibit FGF19 binding to FGFR4 in a competition ELISA with an IC50 of 10 nM, and had no cross reactivity with FGFR1–3. CVX-63 has a beta half life of 55 hrs in the mouse. The anti-tumor activity of CVX-63 was demonstrated in a staged Colo-205 xenograft model, with CVX-63 administered i.v. weekly. Maximal activity of 30% tumor growth inhibition was seen at dosing 10 mg/kg. Immunohistochemistry analysis of treated tumors shows a >90% reduction in phospho-ERK and 50% downregulation of FGFR4 on tumor cells. Ki67, a marker of proliferation was reduced, but active caspase 3 was not found in treated tumors. A patient-derived HCC xenograft was identified which expresses both FGFR4 and FGF19. When tested in this HCC model, CVX-63 did not show any effect inhibiting tumor growth. These data demonstrate that CVX-63 effectively inhibits FGFR4 activity in xenografts. FGFR4 inhibition has a modest effect on human tumor progression and FGFR4 does not seem to be a critical driver of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-396. doi:10.1158/1538-7445.AM2011-LB-396

2493 Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) are ligands of the endothelial cell receptor Tie2. Ang-1 is essential for maintaining stable vasculature. In remodeling endothelial cells, Ang-2 serves as an endogenous Ang-1 antagonist. Ang-2 promotes the dissociation of pericytes from existing blood vessels, and results in unstable blood vessels, which can proliferate and migrate to form new blood vessels in the presence of VEGF. Ang-2 over-expression in many cancers correlates with more invasive disease and poorer prognosis. CVX-060, a specific Ang-2 binding CovX-Body TM , was produced by fusing a chemically modified pharmacophore to the Fab binding site of a specially designed antibody. CVX-060 blocked Ang-2-Tie2 interaction with an IC 50 of 1nM. The anti-tumor activity of CVX-060 was evaluated in staged Colo-205. Significant reduction in tumor growth ( P P P + CD14 + or CD11b + cell number examined by immunohistochemistry were reduced by 76% in CVX-060 treated tumors compared to the Vehicle group ( P TM CVX-060 can effectively reduce Ang-2 protein level and Tie2 + macrophages inside the tumor mass, and inhibit tumor angiogenesis and growth. Significant combination benefit was also observed with other standard of care agents.

1114 CVX-045 is produced by covalently attaching a TSP-1 mimetic comprising a peptidic sequence and a linker, to the Fab binding site of a proprietary scaffold antibody. CVX-045 possesses the potency of the TSP-1 derived peptide, along with the advantageous PK of an antibody. Anti-tumor activity of CVX-045 was evaluated in human xenograft models alone and in combination with standard chemotherapies and targeted molecules. Tumors were staged to 300-400 mm3 prior to initiation of weekly treatments: CVX-045 (10 mg/kg IV weekly) demonstrated significant anti-angiogenic activity, reducing tumor microvessel density 51 % in A549, 36% in A431, and 36% in HT-29 xenografts. In the HT-29 xenograft model, CVX-045 (30 mg/kg) plus 5-FU (100mg/kg) significantly decreased tumor growth rate compared with Vehicle, CVX-045 or 5-FU alone (p

A18 CVX-045, the product of a chemical fusion of a recombinant humanized monoclonal antibody and two anti-angiogenic peptides, is the first in a unique class of fusion proteins. In single dose pharmacokinetic studies, the elimination half-life of CVX-045 increased as test species increased in size. CVX-045 had the longest half-life (T½) in monkeys (80 hr) followed by the rat (65 hr) and the mouse (50 hr). Anti-tumor activity of CVX-045 was evaluated in A549, A431, and HT-29 human xenograft models. Cells were implanted subcutaneously in female nu/nu mice, and tumors were staged to 300-400 mm3 prior to initiation of weekly treatments. CVX-045 (10 mg/kg) significantly reduced A549 and A43I tumor growth 73% (day 49) and 51 % (day 22), respectively, but was not effective in the HT-29 xenograft (10 or 30 mg/kg). CVX-045 demonstrated significant anti-angiogenic activity, reducing tumor microvessel density 51% in A549, 49% in A431, and 36% in HT-29 xenografts. Co-treatment with CVX-045 (30 mg/kg) plus oral sunitinib (15 mg/kg) decreased HT-29 tumor growth rate significantly (p