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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 in vivo 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. Although there was some consistency in general practice, there were examples of extreme case-by-case differences. The responses and variability are discussed herein. Key development challenges were also identified. Results from this survey emphasize the importance for harmonization of regulatory guidelines for the development of gene-therapy products, while still allowing for case-by-case flexibility in nonclinical toxicology studies. However, the appropriate timing for a harmonized guidance, particularly with a platform that continues to rapidly evolve, remains in question., Graphical Abstract, The European Federation of Pharmaceutical Industries and Associates Gene Therapy Working Group conducted a survey on current practices associated with nonclinical toxicity studies conducted to support the development of in vivo viral vector-delivered gene therapies. Responses provide insight on studies conducted, endpoints evaluated, and challenges faced by biopharmaceutical/biotechnology companies.

The number of gene therapies in development continues to increase, as they represent a novel method to treat, and potentially cure, many diseases. Gene therapies can be conducted with an in vivo or ex vivo approach, to cause gene augmentation, gene suppression, or genomic editing. Adeno-associated viruses are commonly used to deliver gene therapies, but their use is associated with several manufacturing, nonclinical and clinical challenges. As these challenges emerge, regulatory agency expectations continue to evolve. Following administration of rAAV-based gene therapies, nonclinical toxicities may occur, which includes immunogenicity, hepatotoxicity, neurotoxicity, and the potential risks for insertional mutagenesis and subsequent tumorgenicity. The mechanism for these findings and translation into the clinical setting are unclear at this time but have influenced the nonclinical studies that regulatory agencies are increasingly requesting to support clinical trials and marketing authorizations. These evolving regulatory expectations and toxicities, as well as future nonclinical considerations, are discussed herein.

A zymogen-like activated factor X variant (FXa) is being developed for treating acute bleeding conditions. Activated factor V is an essential cofactor to FXa for activating prothrombin to thrombin. Thrombi/emboli formation was observed microscopically in FXa toxicity studies in animals. The objective of this research was to evaluate candidate biomarkers for FXa-induced thrombi/emboli formation to inform safety monitoring and dose-escalation decisions in FXa clinical trials. Effects of intravenous FXa administration on platelets, fibrinogen, activated partial thromboplastin time (aPTT), prothrombin time (PT), D-dimer, tissue factor pathway inhibitor, thrombin : antithrombin complex, antithrombin, and factor V, and protein C (PC) activities were evaluated in mice, rats, and monkeys. Mice had endogenous factor V activity 10× that of monkeys and were overly sensitive to FXa-induced thrombi/emboli formation. In monkeys, decreases in fibrinogen and prolongation in aPTT and PT emerged as potential biomarkers for impending FXa-induced thrombi/emboli formation, based on association of changes with microscopically observable thrombi/emboli (0-97 thrombi/emboli per monkey). PC decreases, measured by a clot-based assay, were also observed. A similar reduction in PC activity, when measured by clot-based assay, was observed in a phase 1 clinical trial. However, an in-vitro experiment with human plasma spiked with increasing concentrations of FXa indicated dose-dependent FXa-induced interference with clot-based assays and no depletion of PC or S by FXa in non-clot-based assays. Nonclinical biomarker studies identified fibrinogen, aPTT and PT as potential biomarkers for monitoring the clinical safety of FXa. Results of clot-based assays with FXa treatment should be interpreted with caution.

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.

Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesterase 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free in vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. In vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18F-isotopologue validated our liver-targeting approach.

FXaI16L is a recombinant human FXa variant which is currently being evaluated in the clinic for treating intracerebral hemorrhage. The aim of our studies is to investigate overall pharmacokinetics, pharmacodynamics, and distribution of FXaI16L in preclinical species, and to understand its potential implication in human. Pharmacokinetics of FXaI16L was examined using active site probes and the results showed that FXaI16L displayed fast clearance, low volume of distribution, and a very short plasma resident time in mice, rats, and monkeys. When pharmacodynamics was examined in monkeys, concentration effects of FXaI16L on shortening of active partial prothrombin time and formation of thrombin-antithrombin complex were observed. Furthermore, biodistribution study was conducted in mice using radiolabeled FXaI16L, and showed that 125I-FXaI16L has high plasma protein binding and significant liver and kidney distribution. Human pharmacokinetic prediction for first-in-human dosing was evaluated using allometric scaling, liver blood flow, and a fixed coefficient method, and single species allometric scaling using monkey data was most predictive for human pharmacokinetics of FXaI16L.

This paper presents a systemic investigation of ADA development and ADA impact of a human coagulation factor in nonclinical species during drug development and provides insights into potential implications in human if a similar ADA occurs. FXaI16L-induced ADA response was characterized in monkey, mouse, rat, and dog in different studies, and ADA effects on pharmacokinetic and/or pharmacodynamics of FXaI16L were further examined in ADA-negative and ADA-positive animals. After repeated administrations, FXaI16L elicited a dose and exposure day-dependent ADA response which ranged from no response to a transient or persistent response. Increase in exposure day and increase in dose generally enhanced ADA incidence except for a decrease in ADA incidence was observed in monkeys after repeated high-dose administrations. The observable ADA impact on pharmacokinetics was only found in some ADA+ animals and included decrease in clearance and increase in systemic exposure but no increase in half-life. In addition, no or limited effect on pharmacodynamics by ADA was observed. The earliest ADA response was observed after three exposure days, marked elevation of drug exposure was observed in some animals at log titer > 2.0, and the highest antibody titer excited was about 4 (Log10) in all species. A correlation between ADA induction and accumulative exposure after various repeat treatments in different species was found for FXaI16L. In addition, potential immunogenicity risk and mitigation of ADA in clinics are discussed.

Introduction PF-06438179, a potential biosimilar to Remicade® (infliximab, Janssen Biotech, Inc.), is a chimeric mouse–human monoclonal antibody targeting human tumor necrosis factor alpha (TNF). Methods Analytical (small subset reported here) and nonclinical studies compared the structural, functional, and in vivo nonclinical similarity of PF-06438179 with Remicade sourced from the United States (infliximab-US) and/or European Union (infliximab-EU). Results The peptide map profiles were superimposable, and peptide masses were the same, indicating identical amino acid sequences. Data on post-translational modifications, biochemical properties, and biological function provided strong support for analytical similarity. Administration of a single intravenous (IV) dose (10 or 50 mg/kg) of PF-06438179 or infliximab-EU to male rats was well tolerated. There were no test article-related clinical signs or effects on body weight or food consumption. Systemic exposures [maximum drug concentration (C max) and area under the concentration–time curve (AUC)] in rats administered PF-06438179 or infliximab-EU were similar, with mean exposure ratio of PF-06438179 relative to infliximab-EU ranging from 0.88 to 1.16. No rats developed anti-drug antibodies. A 2-week IV toxicity study was conducted with once-weekly administration of 10 or 50 mg/kg of PF-06438179 to male and female rats. PF-06438179-related hyperplasia of sinusoidal cells occurred in the liver in rats administered 50 mg/kg, but was not adverse based on its minimal to mild severity. The no-observed adverse-effect level for PF-06438179 was 50 mg/kg. At this dose, C max was 1360 µg/mL and AUC at 168 h was 115,000 µg h/mL on day 8. Conclusions The analytical and nonclinical studies have supported advancement of PF-06438179 into global comparative clinical trials. Funding Pfizer Inc.

The contribution of animal testing in drug development has been widely debated and challenged. An industry-wide nonclinical to clinical translational database was created to determine how safety assessments in animal models translate to First-In-Human clinical risk. The blinded database was composed of 182 molecules and contained animal toxicology data coupled with clinical observations from phase I human studies. Animal and clinical data were categorized by organ system and correlations determined. The 2×2 contingency table (true positive, false positive, true negative, false negative) was used for statistical analysis. Sensitivity was 48% with a 43% positive predictive value (PPV). The nonhuman primate had the strongest performance in predicting adverse effects, especially for gastrointestinal and nervous system categories. When the same target organ was identified in both the rodent and nonrodent, the PPV increased. Specificity was 84% with an 86% negative predictive value (NPV). The beagle dog had the strongest performance in predicting an absence of clinical adverse effects. If no target organ toxicity was observed in either test species, the NPV increased. While nonclinical studies can demonstrate great value in the PPV for certain species and organ categories, the NPV was the stronger predictive performance measure across test species and target organs indicating that an absence of toxicity in animal studies strongly predicts a similar outcome in the clinic. These results support the current regulatory paradigm of animal testing in supporting safe entry to clinical trials and provide context for emerging alternate models.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts., Author summary Many disease-mediating proteins have proven difficult to target with traditional small-molecule pharmaceuticals. In this paper, we report that a small molecule, PF-06446846, directly inhibits translation of one such protein, proprotein convertase subtilisin/kexin type 9 (PCSK9), by acting on the translating human ribosome. PF-06446846 causes the translating ribosome to stall soon after translating the PCSK9 signal sequence. We further show that PF-06446846 activity is dependent on the amino acid sequence of the nascent chain inside the ribosome exit tunnel. In a rat safety study, we observe decreases in plasma PCSK9, total cholesterol, and low-density lipoprotein (LDL) cholesterol. Using mass spectrometry in cell culture and ribosome profiling, we demonstrate that despite acting on the ribosome, which synthesizes every protein in the cell, PF-06446846 displays a high level of selectivity for PCSK9. This unexpected potential for small molecules to selectively inhibit the human ribosome opens the possibility for future development of small molecules targeting disease-mediating proteins that were previously thought to be undruggable.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a key role in regulating the levels of plasma low density lipoprotein cholesterol (LDL-C). Here we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome, which allows small molecules to specifically block translation of individual transcripts.One Sentence SummaryA small-molecule PCSK9 inhibitor targets the human ribosome and selectively prevents PCSK9 synthesis.

The ‘totality-of-the-evidence’ biosimilarity concept requires that sufficient structural, functional, nonclinical, and clinical data are acquired in a stepwise manner, to demonstrate that no clinically meaningful differences in quality, safety, or efficacy are observed compared with the reference product. We describe the totality of the evidence for PF-06438179/GP1111 (PF-SZ-IFX; IXIFI™ [infliximab-qbtx]/Zessly®) that supported its approval as an infliximab (IFX) biosimilar for all eligible indications of reference IFX (ref-IFX; Remicade®) in Europe and in the US. Analytical similarity involving in vitro assays capable of distinguishing structural or functional differences between PF-SZ-IFX and ref-IFX formed a foundation for the biosimilarity exercise. Differences identified in N-glycosylation and charge heterogeneity were found not to impact the results in in vitro biological assays reflective of the pharmacology underlying the mechanisms of action (tumor necrosis factor binding, reverse signaling, antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity) of IFX across disease indications. Similarity was assessed in a comparative clinical pharmacokinetic study and in a clinical efficacy and safety study in patients with rheumatoid arthritis, where therapeutic equivalence between PF-SZ-IFX and ref-IFX provided confirmatory evidence of biosimilarity, and, when coupled with the analytical similarity already established, supported extrapolation to all eligible disease indications of ref-IFX.

The cytotoxic and carcinogenic mycotoxin aflatoxin (AF) B1 (AFB1) is biotransformed by the cytochrome P450 monooxygenases (CYP) to a number of relatively nontoxic metabolites, as well as to the ultimate toxic metabolite, AFB1-8,9-epoxide. In a number of tissues and species, AFB1 hydroxylation to the relatively nontoxic metabolite, AFM1, is induced by beta-naphthoflavone (BNF) treatment. Although the liver is the principle target organ for AFB1 toxicity, the mycotoxin is also toxic and carcinogenic to respiratory tissues. To determine if BNF treatment alters the extent of pulmonary AFB1 bioactivation by enhancing detoxification and thereby decreasing epoxidation, the effects of BNF on pulmonary AFB1 metabolism were examined. Rabbit lung cells, isolated by protease digestion and centrifugal elutriation, were incubated with [3H]AFB1. In nonciliated bronchiolar epithelial (Clara) cell-enriched (45-50%) fractions, [3H]AFM1 production (pmol/mg DNA per 2 h) was increased by prior treatment of rabbits with BNF (80 mg/kg per day, 3 and 2 days before cell isolation) as follows: with 1.0 microM [3H]AFB1; control, 10.6 +/- 2.3; BNF, 30.0 +/- 6.4; with 0.10 microM [3H]AFB1; control, 9.4 +/- 4.7; BNF, 20.6 +/- 5.9. With 1.0 microM [3H]AFB1, prior treatment of animals with BNF abolished formation of [3H]aflatoxicol (AFL) but not [3H]AFQ1. The activation (epoxidation) of [3H]AFB1 was measured indirectly as covalent binding to endogenous DNA. With 1.0 microM [3H]AFB1, treatment of rabbits with BNF did not alter DNA binding (pmol/mg DNA per 2 h) in the Clara cell-enriched fraction: control, 103 +/- 41; BNF, 114 +/- 49. However, with 0.10 microM [3H]AFB1, DNA binding in the same fraction was 47% lower in cells from BNF treated animals: control, 17.4 +/- 4.2; BNF, 9.3 +/- 3.9. Formation of 8,9-dihydro-8,9-dihydroxy-AFB1, and the glutathione conjugate of the aflatoxin epoxide (AFB1-GSH) were not detectable at the AFB1 concentration and time point studied, in cells from either BNF-treated or control rabbits. Incubation of isolated, unseparated lung cells from untreated rabbits with 5.0 to 50 microM BNF decreased [3H]AFB1-DNA binding in the presence of 0.1 microM [3H]AFB1 by 35 to 77%, while lower BNF concentrations did not alter DNA binding. In lung cells isolated from BNF treated rabbits, BNF was not detectable (i.e.0.5 microM detection limit). Therefore, the amount of BNF present in isolated rabbit lung cells following in vivo treatment with BNF was below that required to directly inhibit AFB1-DNA adduct formation. The decrease in AFB1-DNA binding from rabbits treated with BNF is apparently due to the selective induction of CYP isozymes and related increases in AFM1 formation, and not to direct inhibition of epoxidation or enhanced conjugation of AFB1-8,9-epoxide with glutathione.

FXaI16L is a variant of activated coagulation factor Xa in which the conformational transition from zymogen to active protease is impaired. Binding to FVa facilitates its transition to the active conformation, rescues procoagulant activity and is hypothesized to localize FXaI16L hemostatic effect to sites of hemorrhage. In nonclinical toxicology studies, thrombi/emboli were observed in animals administered doses exceeding the projected efficacious dose. To identify a possible biomarker predictive of FXaI16L -related thrombi/emboli, effects of FXaI16L on platelets (PLAT), fibrinogen (FBGN), activated partial thromboplastin time (aPTT), prothrombin time (PT), factor V activity (FV), protein C (PROT C) activity, tissue factor pathway inhibitor (TFPI), and antithrombin III (AT-III), were evaluated in in male cynomolgus monkeys (3/group) given FXaI16L for 5 days at IV bolus dosages of 0, 0.1, or 1 mg/kg/day (administered as 0, 0.05, 0.5 mg/kg/dose; BID, 8 h apart). Changes in these parameters were evaluated 5 and 30 min after each dose on Days 1, 3 and 5, and histological evaluation for thrombi/emboli occurred approximately 45 min after first daily dose on Day 5. A single thrombus was observed in the heart of a single animal given 0.1 mg/kg/day, multiple thrombi/emboli were observed in the heart, lung and/or kidney of all animals given 1 mg/kg/day. Compared with pretest values, decreased (range) PLAT (24%-45%), FBGN (11%-86%), aPTT (11%-30%), FV (11%-76%), PROT C (60%-100%), ATIII (2%-26%), and prolonged PT (5%-524%) were observed at ³0.1 mg/kg/day, and prolonged aPTT (17%-367%) and increased TFPI (10%-49%) occurred at 1 mg/kg/day. PROT C activity was below detectable levels 5 min after dosing 1 mg/kg/day, a dose that caused multiple thrombi/emboli. Thus, PROT C activity (as measured by StaClot activity assay) may have utility as a safety biomarker for monitoring treatment with FXaI16L. Depletion of PROT C activity in the 5-day biomarker study in monkeys may reflect actual depletion of PROT C, or may reflect pharmacologic activity of FXaI16L that interferes with the clot based endpoint of this assay since the compound may inherently decrease clotting times. To distinguish between these possibilities, in vitro experiments were performed wherein varying concentrations of FXaI16L were added to normal human plasma and specimens were assayed by the StaClot® (clot based)PROT C activity assay, the StaChrome® (chromogenic) PROT C activity assay and by the Asserachrom® (ELISA) PROT C assay. In a similar manner, effect of FXaI16L on protein S levels was assessed using the StaClot® (clot based) protein S activity assay and an antigenic assay for free protein S. Following in vitro incubation of human plasma with increasing concentrations of FXaI16L, clot-based methods for PROT C and Protein S showed dose-dependent decreases in activity. In contrast, chromogenic and ELISA-based methods for PROT C or antigenic assay for Protein S remained unchanged from baseline levels, confirming that FXaI16L interferes with the StaClot® assay and does not deplete Protein S or PROT C. Abstract 4237. Table 1Comparison of PROT C and Protein S in Human Plasma When Incubated In Vitro with FXaI16L and Assessed by Clot-based or Antigenic AssaysAssay NameIncreasing Concentrations of FXaI16L0 ng/mL1 ng/mL5 ng/mL25 ng/mL50 ng/mL100 ng/mLStaClot PROT Ca137.5 ± 17.37132.8 ± 19.75107.5 ± 24.3457.0 ± 31.01*27.0 ± 19.82*5.3 ± 6.18*Stachrom PROT C122.0 ± 22.73121.3 ± 20.48121.0 ± 22.05121.5 ± 22.41121.5 ± 23.23119.8 ± 21.70Asserachrom PROT C85.8 ± 9.1891.8 ± 11.2790.8 ± 13.4389.0 ± 12.1185.3 ± 14.5286.3 ± 14.38StaClot Protein Sa86.4 ± 21.5766.0 ± 19.6514.6 ± 18.50*0.0 ± 0.00*0.0 ± 0.00*0.0 ± 0.00*Liatest Free Protein S103.6 ± 28.34106.6 ± 28.51100.0 ± 26.67103.0 ± 27.06102.2 ± 29.0399.2 ± 25.35 Data presented as Mean ± S.D. n = 5 donor plasmas/drug concentration. aStaClot PROT C or Protein S – clot-based endpoint. *Statistically different from baseline (0 ng/mL). P Conclusion: Pharmacologic activity of FXaI16L interferes with the clot-based PROT C and protein S assays. However, because marked decreases in the clot-based PROT C assay correlated with the observation of thrombi/emboli in monkeys, this assay remains a useful safety biomarker for monitoring treatment with FXaI16L. Regardless, the impact of drug pharmacology on the respective assay mechanism should be considered when interpreting results. Disclosures Bolt: Pfizer: Employment. Hua:Pfizer Inc: Employment. Brenneman:Pfizer: Employment. Graves:Pfizer: Employment. Arkin:Pfizer Inc.: Employment. Criswell:Pfizer: Employment.