Applications of Cell-Based Protein Array Technology to Preclinical Safety Assessment of Biological Products.

Off-target evaluation is essential in preclinical safety assessments of novel biotherapeutics, supporting lead molecule selection, endpoint selection in toxicology studies, and regulatory requirements for first-in-human trials. Off-target interaction of a therapeutic antibody and antibody derivatives has been historically assessed via the Tissue Cross-Reactivity (TCR) study, in which the candidate molecule is used as a reagent in immunohistochemistry (IHC) to assess binding of the candidate molecule to a panel of human tissue sections. The TCR approach is limited by the performance of the therapeutic as an IHC reagent, which is often suboptimal to outright infeasible.

Comparative assessment of the transduction efficiency and safety associated with the delivery of AAV9-GFP vector via lumbar puncture to cynomolgus macaques with and without anti-AAV9 pre-existing antibodies.

Administration of AAV-based gene therapies into the intra-cerebrospinal fluid (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 anti-AAV9 antibodies at a 5.

Species-Specific Responses to Human Trampling Indicate Alpine Plant Size Is More Sensitive than Reproduction to Disturbance.

Human disturbance, such as trampling, is an integral component of global change, yet we lack a comprehensive understanding of its effects on alpine ecosystems. Many alpine systems are seeing a rapid increase in recreation and in understudied regions, such as the Coast Mountains of British Columbia, yet disturbance impacts on alpine plants remain unclear. We surveyed disturbed (trail-side) and undisturbed (off-trail) transects along elevational gradients of popular hiking trails in the T'ak't'ak'múy'in tl'a In'inyáxa7n region (Garibaldi Provincial Park), Canada, focusing on dominant shrubs (, ) and graminoids ( spp).

CRISPR-Cas9 Editing of the and Promoters to Treat Sickle Cell Disease.

Background: Sickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease.

Regulatory Consideration for the Nonclinical Safety Assessment of Gene Therapies.

The nonclinical safety assessments for gene therapies are evolving, leveraging over 20 years of experimental and clinical experience. Despite the growing experience with these therapeutics, there are no approved harmonized global regulatory documents for developing gene therapies with only the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) S12 guidance on nonclinical biodistribution currently under discussion. Several health authorities have issued guidance over the last 15 years on the nonclinical safety aspects for gene therapy products, but many of the recommendations are limited to high-level concepts on nonclinical safety aspects or altogether silent on key topics.

Classic and evolving approaches to evaluating cross reactivity of mAb and mAb-like molecules - A survey of industry 2008-2019.

Monoclonal antibodies (mAbs) and mAb derivatives have become mainstay pharmaceutical modalites. A critical assessment is to ascertain the specificity of these molecules prior to human clinical trials. The primary technique for determining specificity has been the immunohistochemistry (IHC)-based "Tissue Cross-Reactivity" (TCR) assay, where the candidate molecule is applied to > 30 tissues to look for unexpected staining.

Industry experiences with immune-mediated findings in biotherapeutic nonclinical toxicology studies.

With the growth of monoclonal antibodies and other proteins as major modalities in the pharmaceutical industry, there has been an increase in pharmacology and toxicity testing of biotherapeutics in animals. Animals frequently mount an immune response to human therapeutic proteins. This can result in asymptomatic anti-drug antibody formation, immune complexes that affect drug disposition and/or organ function such as kidney, cytokine release responses, fatal hypersensitivity, or a range of reactions in between.

Nonclinical Studies that Support Viral Vector-Delivered Gene Therapies: An EFPIA Gene Therapy Working Group Perspective.

Nonclinical development strategies for gene therapies are unique from other modalities. The European Federation of Pharmaceutical Industries and Associates (EFPIA) Gene Therapy Working Group surveyed EFPIA member and nonmember pharmaceutical and biotechnology companies about their current practices for designing and implementing nonclinical toxicology studies to support the development of viral vector-delivered gene therapies. Compiled responses from 17 companies indicated that these studies had some variability in species selection, study-design elements, biodistribution, immunogenicity or genomic insertion assessments, safety pharmacology, and regulatory interactions.

Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider.

Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product.

Current strategies in the non-clinical safety assessment of biologics: New targets, new molecules, new challenges.

Nonclinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these innovative and often complex drugs. Emerging topics in this field were discussed recently at the 2016 Annual US BioSafe General Membership meeting. The presentations and subsequent discussions from the main sessions are summarized.

Nonclinical Safety Evaluation of scAAV8- for Treatment of Retinitis Pigmentosa.

Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of deficiency with a self-complementary AAV8 vector carrying the gene for human under control of a short promoter (CPK850). In this article, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation.

Derisking Drug-Induced Carcinogenicity for Novel Therapeutics.

Assessing the carcinogenic potential of innovative drugs spanning diverse therapeutic modalities and target biology represents a major challenge during drug development. Novel modalities, such as cell and gene therapies that involve intrinsic genetic modification of the host genome, require distinct approaches for identification of cancer hazard. We emphasize the need for customized weight-of-evidence cancer risk assessments based on mode of action that balance multiple options for preclinical identification of cancer hazard with appropriate labeling of clinical products and risk management plans.

Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery.

Sensorineural losses of hearing and vestibular sensation due to hair cell dysfunction are among the most common disabilities. Recent preclinical research demonstrates that treatment of the inner ear with a variety of compounds, including gene therapy agents, may elicit regeneration and/or repair of hair cells in animals exposed to ototoxic medications or other insults to the inner ear. Delivery of gene therapy may also offer a means for treatment of hereditary hearing loss.

Inflammation and immunogenicity limit the utility of the rabbit as a nonclinical species for ocular biologic therapeutics.

The nonclinical safety evaluation of therapeutic drug candidates is commonly conducted in two species (rodent and non-rodent) in keeping with international health authority guidance. Biologic drugs typically have restricted species cross-reactivity, necessitating the evaluation of safety in non-human primates and thus limiting the utility of lower order species. Safety studies of cross-reactive ocular biologic drug candidates have been conducted in rabbits as a second toxicology species, despite the fact that rabbits are not a rodent species.

Nonclinical evaluation of GMA161--an antihuman CD16 (FcγRIII) monoclonal antibody for treatment of autoimmune disorders in CD16 transgenic mice.

Fc receptors are a critical component of the innate immune system responsible for the recognition of cross-linked antibodies and the subsequent clearance of pathogens. However, in autoimmune diseases, these receptors play a role in the deleterious action of self-directed antibodies and as such are candidate targets for treatment. GMA161 is an aglycosyl, humanized version of the murine antibody 3G8 that targets the human low-affinity Fcγ receptor III (CD16).

Preclinical safety evaluation of AAV2-sFLT01- a gene therapy for age-related macular degeneration.

AAV2-sFLT01 is a vector that expresses a modified soluble Flt1 receptor designed to neutralize the proangiogenic activities of vascular endothelial growth factor (VEGF) for treatment of age-related macular degeneration (AMD) via an intravitreal injection. Owing to minimal data available for the intravitreal route of administration for adeno-associated virus (AAV), we initiated a 12-month safety study of AAV2-sFLT01 administered intravitreally at doses of 2.4 × 10(9) vector genomes (vg) and 2.

Inhibition of choroidal neovascularization in a nonhuman primate model by intravitreal administration of an AAV2 vector expressing a novel anti-VEGF molecule.

Inhibition of vascular endothelial growth factor (VEGF) for the management of the pathological ocular neovascularization associated with diseases such as neovascular age-related macular degeneration is a proven paradigm; however, monthly intravitreal injections are required for optimal treatment. We have previously shown that a novel, secreted anti-VEGF molecule sFLT01 delivered by intravitreal injection of an AAV2 vector (AAV2-sFLT01) gives persistent expression and is efficacious in a murine model of retinal neovascularization. In the present study, we investigate transduction and efficacy of an intravitreally administered AAV2-sFLT01 in a nonhuman primate (NHP) model of choroidal neovascularization (CNV).

Use of tissue cross-reactivity studies in the development of antibody-based biopharmaceuticals: history, experience, methodology, and future directions.

Tissue cross-reactivity (TCR) studies are screening assays recommended for antibody and antibody-like molecules that contain a complementarity-determining region (CDR), primarily to identify off-target binding and, secondarily, to identify sites of on-target binding that were not previously identified. At the present time, TCR studies involve the ex vivo immunohistochemical (IHC) staining of a panel of frozen tissues from humans and animals, are conducted prior to dosing humans, and results are filed with the initial IND/CTA to support first-in-human clinical trials. In some cases, a robust TCR assay cannot be developed, and in these cases the lack of a TCR assay should not prevent a program from moving forward.

Insulin-like growth factor factor binding protein-2 is a novel mediator of p53 inhibition of insulin-like growth factor signaling.

The p53 tumor suppressor induces cellular growth arrest and apoptosis in response to DNA damage by transcriptionally activating or repressing target genes and also through protein-protein interactions and direct mitochondrial activities. In 1995, insulin-like growth factor binding protein (IGFBP)-3 was identified as one of the genes transcriptionally activated by p53. IGFBP-3 is one of six closely related IGFBP's, with additional IGFBP-related proteins belonging to the IGFBP superfamily.