Short interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) are the most clinically advanced oligonucleotide-based platforms. A number of N-acetylgalactosamine (GalNAc)-conjugated siRNAs (GalNAc-siRNAs), also referred to as RNA interference (RNAi) therapeutics, are currently in various stages of development, though none is yet approved. While the safety of ASOs has been the subject of extensive review, the nonclinical safety profiles of GalNAc-siRNAs have not been reported. With the exception of sequence differences that confer target RNA specificity, GalNAc-siRNAs are largely chemically uniform, containing limited number of phosphorothioate linkages, and 2'-O-methyl and 2'-deoxy-2'-fluoro ribose modifications. Here, we present the outcomes of short-term (3-5 week) rat and monkey weekly repeat-dose toxicology studies of six Enhanced Stabilization Chemistry GalNAc-siRNAs currently in clinical development. In nonclinical studies at supratherapeutic doses, these molecules share similar safety signals, with histologic findings in the organ of pharmacodynamic effect (liver), the organ of elimination (kidney), and the reticuloendothelial system (lymph nodes). The majority of these changes are nonadverse, partially to completely reversible, correlate well with pharmacokinetic parameters and tissue distribution, and often reflect drug accumulation. Furthermore, all GalNAc-siRNAs tested to date have been negative in genotoxicity and safety pharmacology studies.

Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines.

OBJECTIVE:The purpose of this study was to assess the effect of interleukin (IL)-13 deficiency on fertility and reproductive performance of adult mice and on morphological and behavioral development of the offspring.METHODS:Wild-type and homozygous IL-13-deficient (KO) mice were grouped by genotype, and male and female mice were mated within each group. Adult (F(0) ) mice were evaluated for reproductive performance, and development was assessed in F(1) fetuses on gestation day 18, and in F(1) pups to postnatal day 35.RESULTS:In F(0) males, there were no differences in the number of males that mated or impregnated females, or in total sperm count or sperm motility, between the wild-type and KO groups. In F(0) females, there were no observed genotype-related differences in fertility, length of gestation, number of viable fetuses per litter, or viability of offspring. There were no differences in embryo-fetal development (external/palate, skeletal, visceral) of the F(1) fetuses between genotypes. Similarly, IL-13 deficiency had no impact on any postnatal parameters assessed including reflex, sexual maturation, learning, and memory.CONCLUSIONS:IL-13 deficiency had no observed effect on reproductive performance or morphological and behavioral development in mice.

Nature Reviews Immunology 31 Aug 2012 (doi:10.1038/nri3192-c1) On page 2 of the original Correspondence article, under the subheading “Failure to reconcile discordant preclinical data”, the dose of 5.11A1 administered to HIS mice in the study by Legrand et al. was incorrectly stated as 0.3 mg per kg, when it should have been 0.

BACKGROUND To determine if the fetus was affected by maternal antibodies to BMP-2, the antibody response and developmental effects in fetuses from does immunized against recombinant human BMP-2 were evaluated. METHODS Female New Zealand White rabbits received four intramuscular injections (on premating days 1, 8, 22, and 43 [3 days before mating]) of saline and adjuvant (TiterMax(®) Gold [control]) or recombinant human BMP-2 (2 mg/dose) and adjuvant (treated). On GD 29, fetuses were examined, and maternal and fetal anti-BMP-2 titer levels and neutralizing activity were assessed. RESULTS Anti-BMP-2 antibodies were detected in 17 of 18 treated does (127 of 151 fetuses), and low levels were detected in 2 of 16 control does (no fetal exposure observed). In general, levels of fetal anti-BMP-2 antibodies were similar to those in the does, and pregnancy did not boost the immune response to BMP-2. There were no effects of immunization or anti-BMP-2 antibody titer levels on embryo-fetal viability, fetal weight, or fetal external, visceral, or skeletal development. Only a small number of fetuses (n = 4) displayed detectable neutralizing anti-BMP-2 antibodies, but there were no treatment-related effects in those fetuses. CONCLUSIONS The lack of embryo-fetal effects may be due to dosage effects of neutralizing anti-BMP-2 antibodies, timing of exposure (stage and duration) to neutralizing anti-BMP-2 antibodies, and/or redundancy of effects of the various BMPs.

Anti-Aßactive immunization has been proposed as a therapeutic approach for Alzheimer'sdisease (AD). Clinical studies where AD patients were immunized with full-length Aß1-42(AN1792) have resulted in reductions in brain Aß found at autopsy in those patients who had an immune response to the vaccine. These findings have led to the design of additional constructs now undergoing further clinical development. The results of the AN1792 phase 2 clinical study include the observation that approximately 6% of the active treatment group developed signs of meningoencephalitis. Evidence suggests that the observed meningoencephalitis was caused by an Aß directed cytotoxic T-cell response. In an effort to avoid Aß directed T-cell activation, an active vaccine utilizing a short Aß fragment conjugated to a protein carrier (ACC-001) has been developed and is currently undergoing Phase 2 clinical testing. Due, in part, to a favorable NTB efficacy signals seen in antibody responders in the AN1792 phase 2 study and the immunodominant nature of the N-terminus of the Aßpeptide, we chose to focus on the first 7 amino acids of the Aß N-terminal sequence (DAEFRHD). This sequence was conjugated to a mutant diphtheria toxin carrier (CRM197) and was developed as an active vaccine construct. By limiting the Aß sequence to 7 amino acids, it was hypothesized that this vaccine construct would generate relevant anti-Aß antibody responses with no accompanying anti-Aß T-cell response. Non-human primates were injected with vaccine alone or with vaccine plus QS21 adjuvant to evaluate relevant anti-Aß titers and anti-Aß T-cell responses by Elispot. Immunization of non-human primates with ACC-001+QS21adjuvant resulted in measurable anti-Aß titers with no indication of anti-Aß T-cell activity. Anti-Aßactive immunization has been demonstrated to inhibit plaque formation and associated pathology in animal models of Alzheimer's disease. In the AN1792 human clinical trials, active immunization with the full length Aß42peptide sequence resulted in evidence of plaque removal in antibody responders. This approach, however, was limited by the occurrence of meningoencephalitis likely mediated by Aß directed T-cell responses. The current studies detail the development ofACC-001, a 7 amino acid Aßpeptide conjugate vaccine currently in Phase 2 development for the treatment of Alzheimer's disease. Active immunization in non-human primates has demonstrated that this vaccine produces an anti-Aß antibody response similar to full-length Aß immunization, but doesn't appear to generate Aß directed T-cell responses.

Background: Selective neutralization of the IL21/IL21R signaling pathway is a promising approach for the treatment of a variety of autoimmune diseases. Ab-01 is a human neutralizing anti-IL21R antibody. In order to ensure that the activities of Ab-01 are restricted to neutralization even under in vitro cross-linking and in vivo conditions, a comprehensive assessment of agonistic potential of Ab-01 was undertaken. Methods: In vitro antibody cross-linking and cell culture protocols reported for studies with a human agonistic antibody, TGN1412, were followed for Ab-01. rhIL21, the agonist ligand of the targeted receptor, and cross-linked antiCD28 were used as positive controls for signal transduction. In vivo agonistic potential of Ab-01 was assessed by measuring expression levels of cytokine storm-associated and IL21 pathway genes in blood of cynomolgus monkeys before and after IV administration of Ab-01. Results: Using a comprehensive set of assays that detected multiple activation signals in the presence of the positive control agonists, in vitro Ab-01-dependent activation was not detected in either PBMCs or the rhIL21-responsive cell line Daudi. Furthermore, no difference in gene expression levels was detected in blood before and after in vivo Ab-01 dosing of cynomolgus monkeys. Conclusions: Despite efforts to intentionally force an agonistic signal from Ab-01, none could be detected.