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.

Outcome of pediatric patients with acute lymphoblastic leukemia/lymphoblastic lymphoma with hypersensitivity to pegaspargase treated with PEGylated Erwinia asparaginase, pegcrisantaspase: A report from the Children's Oncology Group.

BACKGROUND: Erwinia asparaginase is a Food and Drug Administration approved agent for the treatment of acute lymphoblastic leukemia (ALL) for patients who develop hypersensitivity to Escherichia coli derived asparaginases. Erwinia asparaginase is efficacious, but has a short half-life, requiring six doses to replace one dose of the most commonly used first-line asparaginase, pegaspargase, a polyethylene glycol (PEG) conjugated E. coli asparaginase. Pegcristantaspase, a recombinant PEGylated Erwinia asparaginase with improved pharmacokinetics, was developed for patients with hypersensitivity to pegaspargase. Here, we report a series of patients treated on a pediatric phase 2 trial of pegcrisantaspase. PROCEDURE: Pediatric patients with ALL or lymphoblastic lymphoma and hypersensitivity to pegaspargase enrolled on Children's Oncology Group trial AALL1421 (Jazz 13-011) and received intravenous pegcrisantaspase. Serum asparaginase activity (SAA) was monitored before and after dosing; immunogenicity assays were performed for antiasparaginase and anti-PEG antibodies and complement activation was evaluated. RESULTS: Three of the four treated patients experienced hypersensitivity to pegcrisantaspase manifested as clinical hypersensitivity reactions or rapid clearance of SAA. Immunogenicity assays demonstrated the presence of anti-PEG immunoglobulin G antibodies in all three hypersensitive patients, indicating a PEG-mediated immune response. CONCLUSIONS: This small series of patients, nonetheless, provides data, suggesting preexisting immunogenicity against the PEG moiety of pegaspargase and poses the question as to whether PEGylation may be an effective strategy to optimize Erwinia asparaginase administration. Further study of larger cohorts is needed to determine the incidence of preexisting antibodies against PEG-mediated hypersensitivity to pegaspargase.

Why Do Promising Therapies Stall in Development and How Can We Move Them Forward?

There are many reasons that molecules fail to progress to market and various principles of risk-benefit decisions that can help drive the molecule through development. This symposium included discussions on global strategies involved in pushing promising molecules to market, what to do when a molecule stalls in its progress to market, and options for rescuing the molecule and pushing it forward again. Innovative partnerships that bring stalled drugs back into clinical development were also addressed. A regulatory perspective on common reasons for a molecule to fail in its forward progress was presented. In addition, situations arise when a third-party advisory committee can provide input to help overcome issues identified by a regulatory agency. Using examples from the private and public domain, presentations centered on how to repurpose a molecule and when more science is needed.

Workshop Proceedings: Streamlined Development of Safety Assessment Programs Supporting Orphan/Rare Diseases-Are We There Yet?

A workshop entitled "Streamlined Development of Safety Assessment Programs Supporting Orphan/Rare Diseases-Are We There Yet?" was held at the 36th Annual Meeting of the American College of Toxicology in Summerlin, Nevada. The workshop was sponsored by Shire and Ultragenyx and was designed to present the nonclinical considerations for the development of various products for rare diseases. A panel of experts from industry and government highlighted the nonclinical considerations in developing toxicology programs supporting rare disease therapeutics, challenges in preclinical safety assessment, reviewed the current guidance, and presented the progress that has been made to date. The main learning from the workshop was that nonclinical testing of therapeutics targeting rare disease warrants special considerations, and early collaboration between sponsors and health authorities may help optimize the scope and timing of the supportive studies. Specific examples for nonclinical development programs for enzyme replacement therapy (ERT) were presented. Although the symposium focused on ERTs, the concepts are broadly applicable.

Balancing Efficacy and Safety of an Anti-DLL4 Antibody through Pharmacokinetic Modulation.

PURPOSE: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody. EXPERIMENTAL DESIGN: The F(ab')2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab')2) was generated and assessed in efficacy and toxicity studies. RESULTS: Anti-DLL4 F(ab')2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab')2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition. CONCLUSIONS: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab')2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans.

Phosphorous dysregulation induced by MEK small molecule inhibitors in the rat involves blockade of FGF-23 signaling in the kidney.

MEK, a kinase downstream of Ras and Raf oncogenes, constitutes a high priority target in oncology research. MEK small molecule inhibitors cause soft tissue mineralization in rats secondary to serum inorganic phosphorus (iP) elevation, but the molecular mechanism for this toxicity remains undetermined. We performed investigative studies with structurally distinct MEK inhibitors GEN-A and PD325901 (PD-901) in Sprague-Dawley rats. Our data support a mechanism that involves FGF-23 signal blockade in the rat kidney, causing transcriptional upregulation of 25-hydroxyvitamin D(3) 1-alpha-hydroxylase (Cyp27b1), the rate-limiting enzyme in vitamin D activation, and downregulation of 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1), the enzyme that initiates the degradation of the active form of vitamin D. These transcriptional changes increase serum vitamin D levels, which in turn drive the increase in serum iP, leading to soft tissue mineralization in the rat.

Chronic DLL4 blockade induces vascular neoplasms.

Delta-like 4 (DLL4)-mediated Notch signalling has emerged as an attractive target for cancer therapy. However, the potential side effects of blocking this pathway remain uncertain. Here we show that chronic DLL4 blockade causes pathological activation of endothelial cells, disrupts normal organ homeostasis and induces vascular tumours, raising important safety concerns.

Mode-of-action framework for evaluating the relevance of rodent forestomach tumors in cancer risk assessment.

Studies have shown that a majority of known human carcinogens also cause cancer in laboratory animals. The converse, however, is not as well established-known animal carcinogens are not equally predictive of human carcinogenicity. A particularly controversial aspect of interspecies extrapolation is application of rodent forestomach tumor data for predicting cancer risk in humans, given that a human counterpart for the rodent forestomach does not exist. Proliferative lesions in the rodent forestomach may result from a combination of factors related to route-specific tissue irritation and/or unnatural dosing regimens and are less likely to be relevant in evaluating human carcinogenic potential, particularly when tumors are exclusive to the forestomach. We review the comparative functional anatomy, physiology, tumor biology, tissue concordance, and historical regulatory practices in the use of forestomach tumors for cancer risk assessment, examining specific chemical examples. We also propose a standardized mode-of-action approach that combines multiple risk characterization criteria, including relevance to human exposure conditions, physiologically based toxicokinetics, genotoxicity, and comparative/mechanistic toxicology. Forestomach tumors associated with chronic irritation of the forestomach epithelium, particularly those induced by repeated oral gavage dosing, should not form the basis for carcinogenic classification or quantitative cancer potency estimates for humans. Genotoxic chemicals and those that cause tumors at multiple sites, at doses at or below the maximum tolerated dose, and in the absence of forestomach irritation, are more likely to be relevant human carcinogens. Cancer risk assessment that utilizes forestomach tumor data should consider relevant human exposures, systemic bioavailability, tissue dosimetry and concordance.

Evaluation of mercury in urine as an indicator of exposure to low levels of mercury vapor.

We conducted a pooled analysis to investigate the relationship between exposure to elemental mercury in air and resulting urinary mercury levels, specifically at lower air levels relevant for environmental exposures and public health goals (i.e., < 50 microg/m3 down to 1.0 microg/m3). Ten studies reporting paired air and urine mercury data (149 samples total) met criteria for data quality and sufficiency. The log-transformed data set showed a strong correlation between mercury in air and in urine (r = 0.774), although the relationship was best fit by a series of parallel lines with different intercepts for each study R2 = 0.807). Predicted ratios of air to urine mercury levels at 50 microg/m3 air concentration ranged from 1:1 to 1:3, based on the regression line for the studies. Toward the lower end of the data set (i.e., 10 microg/m3), predicted urinary mercury levels encompassed two distinct ranges: values on the order of 20 microg/L and 30-60 microg/L. Extrapolation to 1 microg/m3 resulted in predicted urinary levels of 4-5 and 6-13 microg/L. Higher predicted levels were associated with use of static area air samplers by some studies rather than more accurate personal air samplers. Urinary mercury predictions based primarily on personal air samplers at 1 and 10 microg/m3 are consistent with reported mean (4 microg/L) and upper-bound (20 microg/L) background levels, respectively. Thus, although mercury levels in air and urine are correlated below 50 microg/m3, the impact of airborne mercury levels below 10 microg/m3 is likely to be indistinguishable from background urinary mercury levels.