Preclinical Enzyme Replacement Therapy with a Recombinant ß-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis.

GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal ß-galactosidase (ß-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine ß-Gal fused to the plant lectin subunit B of ricin (mß-Gal:RTB). We show that long-term, bi-weekly systemic injection of mß-Gal:RTB in the ß-Gal-/- mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, ß-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the ß-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.

Pooled analysis of tanezumab efficacy and safety with subgroup analyses of phase III clinical trials in patients with osteoarthritis pain of the knee or hip.

PURPOSE: A pooled analysis was conducted to evaluate tanezumab efficacy and safety in patients with osteoarthritis (OA), including subgroup analyses of at-risk patients with diabetes, severe OA symptoms, and those aged ≥65 years. PATIENTS AND METHODS: Data from phase III placebo-controlled clinical trials of patients with moderate-to-severe OA of the knee or hip were pooled to evaluate tanezumab efficacy (four trials) and safety (nine trials). Patients received intravenous tanezumab, tanezumab plus an oral NSAID (naproxen, celecoxib, or diclofenac), active comparator (naproxen, celecoxib, diclofenac, or oxycodone), or placebo. Efficacy assessments included change from baseline to week 16 in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain and physical function scores, Patient's Global Assessment (PGA) of OA, and percentage of patients with ≥30%, ≥50%, ≥70%, and ≥90% improvement in WOMAC pain. Safety assessments included adverse event (AE) documentation and physical and neurologic examinations. RESULTS: Tanezumab significantly improved all efficacy end points in the overall population. Efficacy in at-risk patient subgroups was similar to the overall population. Incidence of AEs was highest in the tanezumab plus NSAID group and lowest in the placebo group. Incidence of AEs in the tanezumab monotherapy and active comparator groups was similar. Overall incidence of AEs was similar across subgroups. AEs of abnormal peripheral sensation were more frequently reported in tanezumab-treated patients compared with placebo or active comparator. Patients receiving active comparator had a slightly higher incidence of AEs suggestive of postganglionic sympathetic dysfunction. CONCLUSION: Tanezumab consistently provided significant improvement of pain, physical function, and PGA in individuals with OA, including patients with diabetes, severe OA symptoms, or aged ≥65 years. No increased safety risk was observed in at-risk patient subgroups. TRIAL REGISTRATION: NCT00733902, NCT00744471, NCT00830063, NCT00863304, NCT00809354, NCT00864097, NCT00863772, NCT01089725, NCT00985621.

High-throughput imaging method for direct assessment of GM1 ganglioside levels in mammalian cells.

GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid ß-galactosidase (ß-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1(-/-) cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes.

Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel ß-galactosidase:RTB lectin fusion.

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid ß-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid ß-galactosidase enzyme were produced using a plant-based bioproduction platform. ß-gal:RTB and RTB:ß-gal fusion products retained both lectin activity and ß-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived ß-galactosidase. In contrast, plant-derived ß-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native ß-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both ß-gal:RTB and RTB:ß-gal were processed to the ~64kDa "activated" ß-gal form; the RTB lectin was cleaved and rapidly degraded. The activated ß-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of ß-gal:RTB supported significantly greater accumulation of ß-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived ß-gal. These data demonstrate that plant-made ß-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.

Tanezumab reduces osteoarthritic hip pain: results of a randomized, double-blind, placebo-controlled phase III trial.

OBJECTIVE: To compare the efficacy of tanezumab versus placebo for reducing pain and improving physical function in patients with osteoarthritis (OA) of the hip. METHODS: This was a 32-week, randomized, double-blind, placebo-controlled, phase III trial. Patients with baseline Western Ontario and McMaster Universities OA Index (WOMAC) Pain and Physical Function subscale scores of ≥5 and ≥4, respectively, and patient's global assessment of OA as "fair," "poor," or "very poor" were treated at baseline and weeks 8 and 16. Coprimary efficacy end points were change from baseline to week 16 in WOMAC Pain and Physical Function subscales and patient's global assessment, analyzed using analysis of covariance. Adverse events (AEs) were monitored throughout. RESULTS: Patients (n = 621) were randomized 1:1:1:1 to treatment with intravenous tanezumab 2.5 mg, 5 mg, or 10 mg or placebo. Each tanezumab group showed significant improvement for the 3 coprimary end points versus placebo (P ≤ 0.001 for all). AE incidence ranged from 55% to 58% across tanezumab groups versus 44% for placebo. Safety findings were similar to those previously reported. The tanezumab OA clinical program was temporarily placed on hold because of AEs leading to joint replacement. Total joint replacements were reported in 8 patients: 1 in the 10 mg, 2 in the 5 mg, 2 in the 2.5 mg, and 3 in the placebo group. A total of 9 joints were replaced (8 hips [7 index joints] and 1 shoulder). CONCLUSION: Our findings indicate that tanezumab provides superior pain relief and improvement in physical function and patient's global assessment versus placebo in patients with painful hip OA, and is generally well tolerated.

Tanezumab reduces osteoarthritic knee pain: results of a randomized, double-blind, placebo-controlled phase III trial.

UNLABELLED: The objective of this study was to compare the analgesic efficacy of tanezumab versus placebo in patients with osteoarthritis (OA) of the knee. This was a 32-week, randomized, double-blind, placebo-controlled phase III trial (NCT00733902). The patient criteria included diagnosis of OA; Western Ontario and McMaster Universities OA Index (WOMAC) Pain and Physical Function subscale scores of ≥5 and ≥4, respectively; Patient's Global Assessment of Osteoarthritis (PGA) ≥3; and failure of nonopiate pain medications or candidacy for invasive interventions. Patients received 3 intravenous doses of tanezumab (2.5, 5, or 10 mg) or placebo. The co-primary efficacy end points were changes in WOMAC subscales and PGA at week 16. Adverse events were monitored throughout. Overall, 690 patients (61% female) were randomized and treated. Those treated with tanezumab showed significant improvement in the 3 co-primary end points (P ≤ .015 for all). The incidence of adverse events was 55 to 60% for tanezumab-treated patients versus 48% for placebo-treated patients. Joint replacement was reported in 4 patients, 1 in each treatment group; a total of 5 joints were replaced (1 index knee and 4 hips). The tanezumab OA clinical program is currently on clinical hold due to potential adverse reactions leading to joint replacement. PERSPECTIVE: This is the first phase III randomized, controlled trial to demonstrate that nerve growth factor blockade by tanezumab has superior analgesic efficacy in OA of the knee compared with placebo. Tanezumab was well tolerated, and reports of worsening OA and/or joint replacement were evenly distributed across the treatment groups.

Quality assessment of recombinant proteins produced in plants.

Plant-based expression technologies for recombinant proteins have begun to receive acceptance for pharmaceuticals and other commercial markets. Protein products derived from plants offer safer, more cost-effective, and less capital-intensive alternatives to traditional manufacturing systems using microbial fermentation or animal cell culture bioreactors. Moreover, plants are now known to be capable of expressing bioactive proteins from a diverse array of species including animals and humans. Methods development to assess the quality and performance of proteins manufactured in plants are essential to support the QA/QC demands as plant-produced protein products transition to the commercial marketplace. Within the pharmaceutical arena, process validation and acceptance criteria for biological products must comply with the Food and Drug Administration (FDA) and ICH Q6B guidelines in order to initiate the regulatory approval process. Detailed product specifications will also need to be developed and validated for plant-made proteins for the bioenergy, food, chemical synthesis, or research reagent markets.We have, therefore, developed assessment methods for important qualitative and quantitative parameters of the products and the manufacturing methods utilized in plant-based production systems. In this chapter, we describe a number of procedures to validate product identity and characteristics including mass analyses, antibody cross-reactivity, N-terminal sequencing, and bioactivity. We also address methods for routine assessment of yield, recovery, and purity. The methods presented are those developed for the synthesis and recovery of the avian cytokine, chicken interleukin-12 (ChIL-12), produced in the leaves of Nicotiana benthamiana. The ChIL-12 protein used as a model for this chapter includes a C-terminal histidine epitope (HIS-tag) and, thus, these methods may be directly applicable to other HIS-tagged proteins produced in plants. However, the overall strategy presented using the ChIL-12(HIS) example should provide the basis of standard procedures for assessing the quality of other plant-based protein products and manufacturing systems.

Efficient plant-based production of chicken interleukin-12 yields a strong immunostimulatory cytokine.

Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic in mammals. Therapeutic strategies to develop mammalian IL-12 as a vaccine adjuvant/immunomodulator for promoting cellular immunity and establishing a Th1-biased immune response further support the potential value of ChIL-12. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. We have expressed, characterized, and purified biologically active ChIL-12 in plants using a rapid Agrobacterium-mediated tobacco plant-based transient expression system. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of chicken IL-12 (ChIL-12). A histidine 6x tag was used for identity and purification of ChIL-12(His) protein. Our results demonstrated precise cleavage of the endogenous chicken p40 signal peptide in plants as well as addition of N-linked glycans. Biological activity was confirmed in vitro by interferon-gamma secretion of ChIL-12-treated chicken splenocytes. In addition, splenocytes treated with ChIL-12 expressed with or without the His tag demonstrated comparable ChIFN-gamma induction. These studies indicate that plant-based platforms for bioproduction of complex pharmaceutical proteins produce functional ChIL-12 and provide key advantages in safety, scale, and cost-effective platform for veterinary vaccine and therapeutic applications.