Scientific and Regulatory Policy Committee Points to Consider: Nonclinical Research and Development of In Vivo Gene Therapy Products, Emphasizing Adeno-Associated Virus Vectors.

Sequencing of the human genome and numerous advances in molecular techniques have launched the era of genetic medicine. Increasingly precise technologies for genetic modification, manufacturing, and administration of pharmaceutical-grade biologics have proved the viability of in vivo gene therapy (GTx) as a therapeutic modality as shown in several thousand clinical trials and recent approval of several GTx products for treating rare diseases and cancers. In recognition of the rapidly advancing knowledge in this field, the regulatory landscape has evolved considerably to maintain appropriate monitoring of safety concerns associated with this modality. Nonetheless, GTx safety assessment remains complex and is designed on a case-by-case basis that is determined by the disease indication and product attributes. This article describes our current understanding of fundamental biological principles and possible procedures (emphasizing those related to toxicology and toxicologic pathology) needed to support research and development of in vivo GTx products. This article is not intended to provide comprehensive guidance on all GTx modalities but instead provides an overview relevant to in vivo GTx generally by utilizing recombinant adeno-associated virus-based GTx-the most common in vivo GTx platform-to exemplify the main points to be considered in nonclinical research and development of GTx products.

Use of an animal model of disease for toxicology enables identification of a juvenile no observed adverse effect level for cyclocreatine in creatine transporter deficiency.

In standard general toxicology studies in two species to support clinical development, cyclocreatine, a creatine analog for the treatment of creatine transporter deficiency, caused deaths, convulsions, and/or multi-organ pathology. The potential translatability of these findings to patients was evaluated by comparing toxicity of cyclocreatine in wild-type mice to creatine transporter-deficient mice, a model of the human disease. A biodistribution study indicated greater accumulation of cyclocreatine in the brains of wild-type mice, consistent with its ability to be transported by the creatine transporter. Subsequent toxicology studies confirmed greater sensitivity of wild-type mice to cyclocreatine-induced toxicity. Exposure at the no observed adverse effect level in creatine transporter-deficient (554 µg*hr/ml) mice exceeded exposure at the maximum tolerated dose in wild-type (248 µg*hr/ml) mice. When dosed at 300 mg/kg/day for 3 months, cyclocreatine-related mortality, convulsions, and multi-organ pathology were observed in wild-type mice whereas there were no adverse findings in creatine transporter-deficient mice. Brain vacuolation was common to both strains. Although transporter-deficient mice appeared to be more sensitive, the finding had no functional correlates in this strain. The results highlight the importance of considering models of disease for toxicology in cases where they may be relevant to assessing safety in the intended patient population.

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. In the last few years, however, non-IHC array-based platforms have emerged that allow for screening 75-80% of the human membrane proteome, indicating a viable alternative and/or addition to the IHC methods. The preclinical sciences subcommittee of the Biotechnology Innovation Organization (BIO), "BioSafe", conducted a survey of 26 BIO member companies to understand current sponsor experience with the IHC and array techniques. In the last ten years, respondents noted they have conducted more than 650 IHC TCR assays, largely on full length mAbs, with varying impacts on programs. Protein/cell arrays have been utilized by almost half of the companies and sponsors are gaining familiarity and comfort with the platform. Initial experience with recent versions of these arrays has been largely positive. While most sponsors are not prepared to eliminate the IHC TCR assay, growing experience with these alternatives allows them to confidently choose other approaches with or without TCR assays.

Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

Applying a weight of evidence approach to the evaluation of developmental toxicity of biopharmaceuticals.

Toxicity studies in pregnant animals are not always necessary for assessing the human risk of developmental toxicity of biopharmaceuticals. The growing experience and information on target biology and molecule-specific pharmacokinetics present a powerful approach to accurately anticipate effects of target engagement by biopharmaceuticals using a weight of evidence approach. The weight of evidence assessment should include all available data including target biology, pharmacokinetics, class effects, genetically modified animals, human mutations, and a thorough literature review. When assimilated, this weight of evidence evaluation may be sufficient to inform risk for specific clinical indications and patient populations. While under current guidance this approach is only applicable for drugs and biologics for oncology, the authors would like to suggest that this approach may also be appropriate for other disease indications. When there is an unacceptable level of uncertainty and a toxicity study in pregnant animals could impact human risk assessment, then such studies should be considered. Determination of appropriate nonclinical species for developmental toxicity studies to inform human risk should consider species-specific limitations, reproductive physiology, and pharmacology of the biopharmaceutical. This paper will provide considerations and examples of the weight of evidence approach to evaluating the human risk of developmental toxicity of biopharmaceuticals.

PEGylated Biopharmaceuticals: Current Experience and Considerations for Nonclinical Development.

PEGylation (the covalent binding of one or more polyethylene glycol molecules to another molecule) is a technology frequently used to improve the half-life and other pharmaceutical or pharmacological properties of proteins, peptides, and aptamers. To date, 11 PEGylated biopharmaceuticals have been approved and there is indication that many more are in nonclinical or clinical development. Adverse effects seen with those in toxicology studies are mostly related to the active part of the drug molecule and not to polyethylene glycol (PEG). In 5 of the 11 approved and 10 of the 17 PEGylated biopharmaceuticals in a 2013 industry survey presented here, cellular vacuolation is histologically observed in toxicology studies in certain organs and tissues. No other effects attributed to PEG alone have been reported. Importantly, vacuolation, which occurs mainly in phagocytes, has not been linked with changes in organ function in these toxicology studies. This article was authored through collaborative efforts of industry toxicologists/nonclinical scientists to address the nonclinical safety of large PEG molecules (>10 kilo Dalton) in PEGylated biopharmaceuticals. The impact of the PEG molecule on overall nonclinical safety assessments of PEGylated biopharmaceuticals is discussed, and toxicological information from a 2013 industry survey on PEGylated biopharmaceuticals under development is summarized. Results will contribute to the database of toxicological information publicly available for PEG and PEGylated biopharmaceuticals.

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. The TCR assay by itself has variable correlation with toxicity or efficacy. Therefore, any findings of interest should be further evaluated and interpreted in the context of the overall pharmacology and safety assessment data package. TCR studies are generally not recommended for surrogate molecules or for comparability assessments in the context of manufacturing/cell line changes. Overall, the design, implementation, and interpretation of TCR studies should follow a case-by-case approach.

The application of ICH S6 to the preclinical safety evaluation of plasma derivative therapeutic products.

The ICH S6 guidance was developed to describe a rational science-based flexible approach to the preclinical evaluation for biotechnology-derived pharmaceutical products. It also suggested that some of the principles described may be suitable for plasma-derived therapeutics. Some of the specific concerns unique to protein-based therapeutics include complexity in structure and potential immunogenicity. S6 has been interpreted by some industry and regulatory authorities, often due to lack of experience with these types of products, as encouraging a broader or more conventional toxicology program similar to that normally conducted for small molecules. The guidance does encourage important and necessary preclinical evaluations but also recognizes the limitations of studies in non-relevant animal species because they are without pharmacological interaction with the biologic. In addition, studies of human proteins are often limited in useful chronic, reproductive and carcinogenic toxicity evaluations by the immunological response in animals. Thus the safety evaluation of biopharmaceuticals and plasma derivatives in animals has limitations that cannot be adequately addressed by the use of testing paradigms used for small molecule pharmaceuticals. S6 focuses evaluations on well-designed studies in relevant species for reasonable time periods to make the best use of available resources and enable clinical trials.

Practical approaches to dose selection for first-in-human clinical trials with novel biopharmaceuticals.

Recent advances in our understanding of disease biology, biomarkers, new therapeutic targets, and innovative modalities have each fueled a dramatic expansion in the development of novel human therapeutics. Many are biotechnology-derived biologics possessing high selectivity and affinity for their intended target; as such they often pose challenges in the development path to approval. One challenge is the selection of the first-in-human (FIH) dose. This process has come under increased scrutiny as a result of a FIH trial with a super-agonist monoclonal antibody (TGN1412), which resulted in significant injury to healthy volunteers. Regulatory agencies have responded with supplemental guidance for the development of novel therapeutics. The intent of this paper is to provide experience-based insight, with relevant examples, for those planning the first administration of novel biopharmaceuticals in humans.

Considerations in assessing the developmental and reproductive toxicity potential of biopharmaceuticals.

This report discusses the principles of developmental and reproductive toxicity (DART) testing for biopharmaceuticals. Biopharmaceuticals are large-molecular-weight proteins or peptides produced by modern biotechnology techniques incorporating genetic engineering and hybridoma technologies. The principles of DART testing for biopharmaceuticals are similar to those for small-molecule pharmaceuticals and in general follow the regulatory guidance outlined in International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) document S5(R2). However, because many biopharmaceuticals are species-specific, alternate approaches may be needed to evaluate DART potential as outlined in ICH S6. For molecules that show species-specific cross-reactivity restricted to non-human primates (NHP), some aspects of DART may require NHP testing. For biopharmaceuticals that are uniquely specific and only active on intended human targets or human and chimpanzee targets, surrogate molecules that cross-react with the more traditional rodent species may need to be developed and used for DART testing. Alternatively, genetically modified transgenic animals may also need to be considered. Surrogate molecules and transgenic animals may also be considered for DART testing even if the biopharmaceutical is active in NHPs in order to reduce the use of NHPs. Because of the unique properties of biopharmaceuticals, a case-by-case approach is needed for DART and general toxicity evaluation, which requires consideration of specific product attributes including biochemical and biophysical characteristics, pharmacological activity, and intended clinical indication.

Toxicity testing of a novel riboflavin-based technology for pathogen reduction and white blood cell inactivation.

The Mirasol PRT System (Gambro BCT, Lakewood, CO) for platelets and plasma uses riboflavin and UV light to reduce pathogens and inactivate white blood cells in donated blood products. An extensive toxicology program, developed in accordance with International Organisation for Standardisation (ISO) 10993 guidelines, was performed for the Mirasol PRT system. Test and control articles for most of the reported studies were treated (test) or untreated (control) blood products. For some studies, pure lumichrome (the major photoproduct of riboflavin) or photolyzed riboflavin solution was used. Systemic toxicity was evaluated with in vivo animal studies in the acute and subchronic settings. Developmental toxicity was evaluated with an in vivo animal study. Genotoxicity and neoantigenicity were evaluated with in vitro and in vivo tests. Hemocompatibility and cytotoxicity were assessed with standard, in vitro assays. The pharmacokinteics, excretion, and tissue distribution of (14)C-riboflavin and its photoproducts was evaluated with an in vivo animal study. The possible presence of leachable or extractable compounds (from the disposable set) was evaluated with novel assays for measuring these compounds in blood. No treatment-related toxicity was observed in any of the studies.

Safety testing of monoclonal antibodies in non-human primates: Case studies highlighting their impact on human risk assessment.

Monoclonal antibodies (mAbs) are improving the quality of life for patients suffering from serious diseases due to their high specificity for their target and low potential for off-target toxicity. The toxicity of mAbs is primarily driven by their pharmacological activity, and therefore safety testing of these drugs prior to clinical testing is performed in species in which the mAb binds and engages the target to a similar extent to that anticipated in humans. For highly human-specific mAbs, this testing often requires the use of non-human primates (NHPs) as relevant species. It has been argued that the value of these NHP studies is limited because most of the adverse events can be predicted from the knowledge of the target, data from transgenic rodents or target-deficient humans, and other sources. However, many of the mAbs currently in development target novel pathways and may comprise novel scaffolds with multi-functional domains; hence, the pharmacological effects and potential safety risks are less predictable. Here, we present a total of 18 case studies, including some of these novel mAbs, with the aim of interrogating the value of NHP safety studies in human risk assessment. These studies have identified mAb candidate molecules and pharmacological pathways with severe safety risks, leading to candidate or target program termination, as well as highlighting that some pathways with theoretical safety concerns are amenable to safe modulation by mAbs. NHP studies have also informed the rational design of safer drug candidates suitable for human testing and informed human clinical trial design (route, dose and regimen, patient inclusion and exclusion criteria and safety monitoring), further protecting the safety of clinical trial participants.

Considerations for the Characterization and Interpretation of Results Related to Alternative Complement Activation in Monkeys Associated with Oligonucleotide-Based Therapeutics.

This article provides an overview of the discussions held by the Immunomodulatory Subcommittee of the Oligonucleotide Safety Working Group on complement activation induced by oligonucleotides, most notably the phosphorothioate-containing oligonucleotides. Alternative complement pathway activation in monkeys is a common effect of single-stranded phosphorothioate backbone oligonucleotides in toxicology studies. This article discusses the mechanism for activation, general investigational strategy, and the impact of various chemical modifications. The goal is to provide the best practice approach to characterizing this effect, understanding the implication of the species specificity, and the interpretation of clinical relevance.

Regulatory acceptance of animal models of disease to support clinical trials of medicines and advanced therapy medicinal products.

The utility of animal models of disease for assessing the safety of novel therapeutic modalities has become an increasingly important topic of discussion as research and development efforts focus on improving the predictive value of animal studies to support accelerated clinical development. Medicines are approved for marketing based upon a determination that their benefits outweigh foreseeable risks in specific indications, specific populations, and at specific dosages and regimens. No medicine is 100% safe. A medicine is less safe if the actual risks are greater than the predicted risks. The purpose of preclinical safety assessment is to understand the potential risks to aid clinical decision-making. Ideally preclinical studies should identify potential adverse effects and design clinical studies that will minimize their occurrence. Most regulatory documents delineate the utilization of conventional "normal" animal species to evaluate the safety risk of new medicines (i.e., new chemical entities and new biological entities). Animal models of human disease are commonly utilized to gain insight into the pathogenesis of disease and to evaluate efficacy but less frequently utilized in preclinical safety assessment. An understanding of the limitations of the animal disease models together with a better understanding of the disease and how toxicity may be impacted by the disease condition should allow for a better prediction of risk in the intended patient population. Importantly, regulatory authorities are becoming more willing to accept and even recommend data from experimental animal disease models that combine efficacy and safety to support clinical development.

Pre-clinical Specificity and Safety of UC-961, a First-In-Class Monoclonal Antibody Targeting ROR1.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen. Because of its expression on the cell surface of leukemia cells from patients with chronic lymphocytic leukemia (CLL), but not on normal B-cells or other postpartum tissues, ROR1 is an attractive candidate for targeted therapies. UC-961 is a first-in-class humanized monoclonal antibody that binds the extracellular domain of ROR1. In this article we outline some of the preclinical studies leading to an investigational new drug designation, enabling clinical studies in patients with CLL.

Considerations for assessment of reproductive and developmental toxicity of oligonucleotide-based therapeutics.

This white paper summarizes the current consensus of the Reproductive Subcommittee of the Oligonucleotide Safety Working Group on strategies to assess potential reproductive and/or developmental toxicities of therapeutic oligonucleotides (ONs). The unique product characteristics of ONs require considerations when planning developmental and reproductive toxicology studies, including (a) chemical characteristics, (b) assessment of intended and unintended mechanism of action, and (c) the optimal exposure, including dosing regimen. Because experience across the various classes of ONs as defined by their chemical backbone is relatively limited, best practices cannot be defined. Rather, points to consider are provided to help in the design of science-based reproductive safety evaluation programs based upon product attributes.

Oligo safety working group exaggerated pharmacology subcommittee consensus document.

This document summarizes the current consensus opinion of the Exaggerated Pharmacology (EP) Subcommittee of the Oligonucleotide Safety Working Group on the appropriate strategies to assess potential adverse effects caused by an "exaggerated" degree of the intended pharmacologic activity of an oligonucleotide (ON). The Subcommittee focused its discussions primarily on the ON subclasses that impact expression of "host" (i.e., human gene products--antisense, small interfering RNAs, and related ONs that target messenger RNA), with later and more limited discussions on aptamer, immunostimulatory, and microRNA subclasses. It is expected that many of these principles will be relevant to other subclasses but will need to be carefully considered as those development programs advance towards clinical trials. The recommendations may also serve as a frame of reference when designing Good Laboratory Practice safety studies with ONs, with regard to the study design elements that address assessment of EP. It is also hoped that these recommendations will establish a foundation for discussion with regulatory agencies on this subject.