Alternatives to Monkey Reproductive Toxicology Testing for Biotherapeutics.

Embryofetal toxicity studies are conducted to support inclusion of women of childbearing potential in clinical trials and to support labeling for the marketed pharmaceutical product. For biopharmaceuticals, which frequently lack activity in the rodent or rabbit, the nonhuman primate is the standard model to evaluate embryofetal toxicity. These studies have become increasingly challenging to conduct due to the small number of facilities capable of performing them and a shortage of sexually mature monkeys. The low number of animals per group and the high rate of spontaneous abortion in cynomolgus monkeys further complicate interpretation of the data. Recent FDA guidance has proposed a weight of evidence (WoE) approach to support product labeling for reproductive toxicity of products intended to be used for the treatment of cancer (Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling Recommendations), an approach that has also supported the approval of biotherapeutics for non-cancer indications. Considerations to determine the appropriateness and content of a WoE approach to support product labeling for embryofetal risk include known class effects in humans; findings from genetically modified animals with or without drug administration; information from surrogate compounds; literature-based assessments about the developmental role of the pharmaceutical target; and the anticipated exposure during embryofetal development. This paper summarizes the content of a session presented at the 42nd annual meeting at the American College of Toxicology, which explored the conditions under which alternative approaches may be appropriate to support product labeling for reproductive risk, and how sponsors can best justify the use of this approach.

Preclinical Safety Assessment and Toxicokinetics of Apitegromab, an Antibody Targeting Proforms of Myostatin for the Treatment of Muscle-Atrophying Disease.

Myostatin is a negative regulator of skeletal muscle and has become a therapeutic target for muscle atrophying disorders. Although previous inhibitors of myostatin offered promising preclinical data, these therapies demonstrated a lack of specificity toward myostatin signaling and have shown limited success in the clinic. Apitegromab is a fully human, monoclonal antibody that binds to human promyostatin and latent myostatin with a high degree of specificity, without binding mature myostatin and other closely related growth factors. To support the clinical development of apitegromab, we present data from a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies confirmed the ability of apitegromab to inhibit the activation of promyostatin. Toxicology studies in monkeys for 4 weeks and in adult rats for up to 26 weeks showed that weekly intravenous administration of apitegromab achieved sustained serum exposure and target engagement and was well-tolerated, with no treatment-related adverse findings at the highest doses tested of up to 100 mg/kg and 300 mg/kg in monkeys and rats, respectively. Additionally, results from an 8-week juvenile rat study showed no adverse effects on any endpoint, including neurodevelopmental, motor, and reproductive outcomes at 300 mg/kg administered weekly IV. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that apitegromab is a selective inhibitor of proforms of myostatin that does not exhibit toxicities observed with other myostatin pathway inhibitors. These data support the conduct of ongoing clinical studies of apitegromab in adult and pediatric patients with spinal muscular atrophy (SMA).

Nonclinical Development of SRK-181: An Anti-Latent TGFß1 Monoclonal Antibody for the Treatment of Locally Advanced or Metastatic Solid Tumors.

Checkpoint inhibitors offer a promising immunotherapy strategy for cancer treatment; however, due to primary or acquired resistance, many patients do not achieve lasting clinical responses. Recently, the transforming growth factor-ß (TGFß) signaling pathway has been identified as a potential target to overcome primary resistance, although the nonselective inhibition of multiple TGFß isoforms has led to dose-limiting cardiotoxicities. SRK-181 is a high-affinity, fully human antibody that selectively binds to latent TGFß1 and inhibits its activation. To support SRK-181 clinical development, we present here a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies showed that SRK-181 has no effect on human platelet function and does not induce cytokine release in human peripheral blood. Four-week toxicology studies with SRK-181 showed that weekly intravenous administration achieved sustained serum exposure and was well tolerated in rats and monkeys, with no treatment-related adverse findings. The no-observed-adverse-effect levels levels were 200 mg/kg in rats and 300 mg/kg in monkeys, the highest doses tested, and provide a nonclinical safety factor of up to 813-fold (based on Cmax) above the phase 1 starting dose of 80 mg every 3 weeks. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that SRK-181 is a selective inhibitor of latent TGFß1 that does not produce the nonclinical toxicities associated with nonselective TGFß inhibition. These data support the initiation and safe conduct of a phase 1 trial with SRK-181 in patients with advanced cancer.

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. The topics covered included: (i) specialty biologics (oncolytic virus, gene therapy, and gene editing-based technologies), (ii) the value of non-human primates (NHPs) for safety assessment, (iii) challenges in the safety assessment of immuno-oncology drugs (T cell-dependent bispecifics, checkpoint inhibitors, and costimulatory agonists), (iv) emerging therapeutic approaches and modalities focused on microbiome, oligonucleotide, messenger ribonucleic acid (mRNA) therapeutics, (v) first in human (FIH) dose selection and the minimum anticipated biological effect level (MABEL), (vi) an update on current regulatory guidelines, International Council for Harmonization (ICH) S1, S3a, S5, S9 and S11 and (vii) breakout sessions that focused on bioanalytical and PK/PD challenges with bispecific antibodies, cytokine release in nonclinical studies, determining adversity and NOAEL for biologics, the value of second species for toxicology assessment and what to do if there is no relevant toxicology species.

Nonclinical Development of ANX005: A Humanized Anti-C1q Antibody for Treatment of Autoimmune and Neurodegenerative Diseases.

ANX005 is a humanized immunoglobulin G4 recombinant antibody against C1q that inhibits its function as the initiating molecule of the classical complement cascade. The safety and tolerability of ANX005 are currently being evaluated in a phase I trial in healthy volunteers ( www.clinicaltrials.gov Identifier: NCT03010046). Inhibition of C1q can be applied therapeutically in a broad spectrum of diseases, including acute antibody-mediated autoimmune disease, such as Guillain-Barré syndrome (GBS), and in chronic diseases of the central nervous system involving complement-mediated neurodegeneration, such as Alzheimer's disease (AD). To support the clinical development of ANX005, several studies were conducted to assess the pharmacology, pharmacokinetics, and potential toxicity of ANX005. ANX-M1, the murine precursor of ANX005, functionally inhibits the classical complement cascade both in vitro and in vivo, to protect against disease pathology in mouse models of GBS and AD. Toxicology studies with ANX005, itself, showed that intravenous administration once weekly for 4 weeks was well tolerated in rats and monkeys, with no treatment-related adverse findings. Serum levels of ANX005 in monkeys correlate with a reduction in free C1q levels both in the serum and in the cerebrospinal fluid. In summary, ANX005 has shown proof of concept in in vitro and in vivo nonclinical pharmacology models, with no toxicity in the 4-week repeat-dose studies in rats and monkeys. The no observed adverse effect level was 200 mg/kg/dose, which is 200-fold higher than the first-in-human starting dose of 1 mg/kg in healthy volunteers.

An Introduction to the Regulatory and Nonclinical Aspects of the Nonclinical Development of Antibody Drug Conjugates.

Antibody drug conjugates (ADCs) are promising therapies currently in development for oncology with unique and challenging regulatory and scientific considerations. While there are currently no regulatory guidelines specific for the nonclinical development of ADCs, there are harmonized international guidelines (e.g., ICHS6(R1), ICHM3(R2), ICHS9) that apply to ADCs and provide a framework for their complex development with issues that apply to both small and large molecules. The regulatory and scientific perspectives on ADCs are evolving due to both the advances in ADC technology and a better understanding of the safety and efficacy of ADCs in clinical development. This paper introduces the key scientific and regulatory aspects of the nonclinical development of ADCs, discusses important regulatory and scientific issues in the nonclinical to clinical dose translation of ADCs, and introduces new concepts in the areas of pharmacokinetic/pharmacodynamic (PK/PD) modeling and simulation.

The development of therapeutic monoclonal antibodies: overview of the nonclinical safety assessment.

Monoclonal antibodies (mAbs) represent a class of biotechnology-derived therapeutics for use in the treatment of various disease indications such as oncology, autoimmune, cardiovascular, and metabolic disorders. Monoclonal antibodies are immunoglobulin (Ig) proteins engineered to bind to specific antigens with high specificity. The concepts reviewed in this paper include 1) the regulatory procedures and guidelines that apply to mAbs, 2) the types of toxicology studies applicable to mAbs, and 3) the scientific challenges, such as the selection of a relevant animal species and the development of anti-drug antibodies, that can arise due to the unique properties of mAbs.

Considerations regarding nonhuman primate use in safety assessment of biopharmaceuticals.

Selection of a pharmacologically responsive species can represent a major challenge in designing nonclinical safety assessment programs for many biopharmaceuticals (eg, monoclonal antibodies (mAbs)). Frequently, the only relevant species for nonclinical testing of mAbs is the non-human primate (NHP). This situation, coupled with a rapidly increasing number of mAb drugs in development, has resulted in a significant increase in the number of NHPs used in nonclinical safety assessment. Apart from ethical considerations related to responsible animal use, there is a clear need for more efficient and innovative approaches to drug discovery and development; these factors drive the need to investigate alternative approaches and strategies for the safety assessment. This review summarizes important scientific and regulatory perspectives derived from presentations and audience discussions in an educational forum at the 2010 annual American College of Toxicology meeting regarding opportunities for employing alternative approaches to minimize NHP use in mAb drug development.

A 14-day repeat dose oral gavage range-finding study of a first-in-class CDI investigational antibiotic, in rats.

Drug resistant bacteria are winning the fight over antibiotics with some bacteria not responding to any antibiotics, threatening modern medicine as we know it. The development of new, effective and safe antibiotics is critical for addressing this issue. Ramizol, a first-in-class styrylbenzene based antibiotic, is an investigational drug indicated for Clostridium difficile infections (CDI). The objective of this range-finding study was to evaluate the potential general toxicity (based on toxicological endpoints selected) and toxicokinetics of Ramizol in male and female rats that may arise from repeated exposure via oral gavage over a test period of at least 14 days at doses of 50 mg/kg, 500 mg/kg and 1500 mg/kg. There were no mortalities in this study and no Ramizol-related clinical observations. Additionally, there were no changes in mean body weight, body weight gain, food consumption or food efficiency for male and female rats attributable to Ramizol administration. The observed pharmacokinetic behavior showed the presence of Ramizol in plasma at 24 hours post-dosing combined with increasing AUC(0-24) values during the course of this study in groups administered 1500 mg/kg/day, which suggests that at least some dosing groups will show accumulation of compound during repeated dose studies. These toxicology results have shown Ramizol is well-tolerated at very high concentrations in rats and support the further drug development of Ramizol as a first-in-class antibiotic for the treatment of CDI.

Biomarkers for nonclinical infusion reactions in marketed biotherapeutics and considerations for study design.

The observation of an infusion reaction (IR) in a nonclinical study can cause concern among investigators and regulators in the development of biotherapeutics. Biomarkers can be informative to determine whether the reactions are immune-mediated or test-article related and if there is a potential risk to human subjects. IRs encompass a broad range of adverse events with a variety of triggers; the focus of this paper is IRs due to cytokine release syndrome or immune complex formation and the associated biomarkers. Such reactions generally do not preclude clinical development or marketing approval, because it is widely accepted that immune-mediated reactions in nonclinical species are not predictive of human outcomes. Several US approved products (from 2004 to 2016) have documented IRs in nonclinical species. This review article discusses recent examples, the biomarkers evaluated, and implications for study design and conduct.

Cosegregation of chromosomes containing immortal DNA strands in cells that cycle with asymmetric stem cell kinetics.

A long-standing intriguing hypothesis in cancer biology is that adult stem cells avoid mutations from DNA replication errors by a unique pattern of chromosome segregation. At each asymmetric cell division, adult stem cells have been postulated to selectively retain a set of chromosomes that contain old template DNA strands (i.e., "immortal DNA strands"). Using cultured cells that cycle with asymmetric cell kinetics, we confirmed both the existence of immortal DNA strands and the cosegregation of chromosomes that bear them. Our findings also lead us to propose a role for immortal DNA strands in tissue aging as well as cancer.