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The objectives of the study were to use tumor size data from 10 phase II/III atezolizumab studies across five solid tumor types to estimate tumor growth inhibition (TGI) metrics and assess the impact of TGI metrics and baseline prognostic factors on overall survival (OS) for each tumor type. TGI metrics were estimated from biexponential models and posttreatment longitudinal data of 6699 patients. TGI‐OS full models were built using parametric survival regression by including all significant baseline covariates from the Cox univariate analysis followed by a backward elimination step. The model performance was evaluated for each trial by 1000 simulations of the OS distributions and hazard ratios (HR) of the atezolizumab‐containing arms versus the respective controls. The tumor growth rate estimate was the most significant predictor of OS across all tumor types. Several baseline prognostic factors, such as inflammatory status (C‐reactive protein, albumin, and/or neutrophil‐to‐lymphocyte ratio), tumor burden (sum of longest diameters, number of metastatic sites, and/or presence of liver metastases), Eastern Cooperative Oncology Group performance status, and lactate dehydrogenase were also highly significant across multiple studies in the final multivariate models. TGI‐OS models adequately described the OS distribution. The model‐predicted HRs indicated good model performance across the 10 studies, with observed HRs within the 95% prediction intervals for all study arms versus controls. Multivariate TGI‐OS models developed for different solid tumor types were able to predict treatment effect with various atezolizumab monotherapy or combination regimens and could be used to support design and analysis of future studies.

The time-varying clearance (CL) of the PD-L1 inhibitor atezolizumab was assessed on a population of 1519 cancer patients (primarily with non-small-cell lung cancer or metastatic urothelial carcinoma) from three clinical studies. The first step was to identify the baseline covariates affecting atezolizumab CL without including time-varying components (stationary covariate model). Two time-varying models were then investigated: (1) a model allowing baseline covariates to vary over time (time-varying covariate model), (2) a model with empirical time-varying Emax CL function. The final stationary covariate model included main effects of body weight, albumin levels, tumor size, anti-drug antibodies (ADA) and gender on atezolizumab CL. Both time-varying models resulted in a clear improvement of the data fit and visual predictive checks over the stationary model. The time-varying covariate model provided the best fit of the data. In this model, the main driver for change in CL over time was variations in albumin level with an increase in serum albumin (improvement in a patient’s status) mirroring a decrease in CL. Time-varying ADAs had a small impact (9% increase in CL). None of the covariates impacted atezolizumab CL by more than ± 30% from median. The estimated maximum decrease in CL with time was 22% with the Emax model. The overall impact of covariates on atezolizumab CL did not warrant any change in atezolizumab dosing recommendations. The results support the hypothesis that variation in atezolizumab CL over time is associated with patients’ disease status, as shown with other checkpoint inhibitors.

Antibody therapeutics can be associated with unwanted immune responses resulting in the development of anti‐drug antibodies (ADA). Optimal methods to evaluate the potential effects of ADA on clinical outcomes in oncology are not well established. In this study, we assessed efficacy and safety, based on ADA status, in patients from over 10 clinical trials that evaluated the immune checkpoint inhibitor atezolizumab as a single agent or as combination therapy for several types of advanced cancers. ADA can only be observed post randomization, and imbalances in baseline prognostic factors can confound the interpretation of ADA impact. We applied methodology to account for the confounding effects of baseline clinical characteristics and survivorship bias on efficacy. Adjusted meta‐analyses revealed that despite numerical differences in overall survival and progression‐free survival between ADA‐positive and ADA‐negative patients from some studies, ADA‐positive patients from studies with an overall treatment effect derived benefit from atezolizumab, compared with their adjusted controls. Based on large, pooled populations from atezolizumab monotherapy or combination studies, unadjusted descriptive analyses did not identify a clear relationship between ADA status and frequency or severity of adverse events. Data also suggested that any ADA impact is not driven by neutralizing activity. Collectively, this exploratory analysis suggests that the potential for ADA development should not impact treatment decisions with atezolizumab.

Baseline patient characteristics and prognostic factors are important considerations in oncology when evaluating the impact of immunogenicity on pharmacokinetics (PK) and efficacy. Here, we assessed the impact of anti‐drug antibodies (ADA) on the PK of the immune checkpoint inhibitor atezolizumab (an anti–PD‐L1 monoclonal antibody). We evaluated data from ≈ 4500 patients from 12 clinical trials across different tumor types, treatment settings, and dosing regimens. In our dataset, ~ 30% of patients (range, 13–54%) developed treatment‐emergent ADA, and in vitro neutralizing antibodies (NAb) were seen in ~ 50% of ADA‐positive (+) patients. Pooled time course data showed a trend toward lower atezolizumab exposure in ADA+ patients, which was more pronounced in ADA+/NAb+ patients. However, the atezolizumab concentration distributions overlapped, and drug concentrations exceeded 6 µg/ml, the target concentration required for receptor saturation, in greater than 95% of patients. Patients had sufficient exposure regardless of ADA status. The dose selected to allow for dosing over effects from ADA resulted in a flat exposure‐response relationship. Analysis of study results by ADA titer showed that exposure and overall survival were not affected in a clinically meaningful way. High tumor burden, low albumin, and high CRP at baseline showed the greatest association with ADA development but not with subsequent NAb development. These imbalanced factors at baseline can confound analysis of ADA impact. ADA increases atezolizumab clearance minimally (9%), and its impact on exposure based on the totality of the clinical pharmacology assessment does not appear to be clinically meaningful.

The myelin sheath insulates axons in the vertebrate nervous system, allowing rapid propagation of action potentials via saltatory conduction. Specialized glial cells, termed Schwann cells in the PNS and oligodendrocytes in the CNS, wrap axons to form myelin, a compacted, multilayered sheath comprising specific proteins and lipids. Disruption of myelinated axons causes human diseases, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. Despite the progress in identifying human disease genes and other mutations disrupting glial development and myelination, many important unanswered questions remain about the mechanisms that coordinate the development of myelinated axons. To address these questions, we began a genetic dissection of myelination in zebrafish. Here we report a genetic screen that identified 13 mutations, which define 10 genes, disrupting the development of myelinated axons. We present the initial characterization of seven of these mutations, defining six different genes, along with additional characterization of mutations that we have described previously. The different mutations affect the PNS, the CNS, or both, and phenotypic analyses indicate that the genes affect a wide range of steps in glial development, from fate specification through terminal differentiation. The analysis of these mutations will advance our understanding of myelination, and the mutants will serve as models of human diseases of myelin.

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.

Publisher Summary This chapter focuses on the downregulation of gene expression with negatively charged peptide nucleic acids (PNAs) in zebrafish embryos. Genetic analyses in the zebrafish Danio rerio have proved quite useful for identifying genes that direct vertebrate development. Several genes necessary for normal embryonic development have been characterized by visual characterization of morphological phenotypes. Uroporphyrinogen decarboxylase, an enzyme essential for heme biosynthesis, is also essential for embryonic development. Mutants for uroD exhibit hepatoerythropoietic porphyria (HEP), which is characterized by excessive accumulation of porphyrin compounds, and, as a result, autofluorescent, photosensitive erythrocytes. The ability to turn off individual genes at will in growing cells provides a powerful tool for elucidating the role of a particular gene, for diagnosis, and for therapeutic intervention. The development of morpholino-based knockdown technology in zebrafish has enabled sequence-based reverse genetic screens. Zebrafish are the most appropriate species for analysis because they are small in size, easy to maintain and breed, and produce large numbers of progeny on a daily basis. Their embryos develop rapidly and are optically clear, permitting direct observation of the developing organs. Negatively charged, highly soluble PNA analogs with alternating phosphonates (HypNA-pPNAs) are effective and specific antisense agents in zebrafish embryos, showing comparable potency and greater specificity against chordin, ntl, and uroD.

Polar pili biogenesis in Caulobacter involves the asymmetric localization of the CpaE and CpaC components of the pili-specific secretion apparatus to one pole of the predivisional cell followed by the biosynthesis of the pili filaments in the daughter swarmer cell. The histidine kinase signaling protein, PleC, that controls the temporal accumulation of the PilA pilin subunit is asymmetrically localized to the pole at which pili are assembled. Here we identify a protein, PodJ, that provides the positional information for the polar localization of both PleC and CpaE. The PodJ protein was found to exist in two forms, a truncated 90-kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. When active PleC is delocalized in a Δ podJ mutant, the accumulation of PilA, the downstream target of PleC signaling, is impaired, providing evidence that the polar localization of this histidine kinase stimulates the response signaled by a two-component system.

Each cell division in Caulobacter crescentus is asymmetric, yielding a swarmer cell with several polar pili and a non-piliated stalked cell. To identify factors contributing to the asymmetric biogenesis of polar pili, cytological studies of pilus assembly components were performed. We show here that the CpaC protein, which is thought to form the outer membrane pilus secretion channel, and its assembly factor, CpaE, are localized to the cell pole prior to the polymerization of the pilus filament. We demonstrate that the PleC histidine kinase, a two-component signal transduction protein shown previously to localize to the piliated cell pole before and during pilus assembly, controls the accumulation of the pilin subunit, PilA. Using an inactive form of PleC (PleCH610A) that lacks the catalytic histidine residue, we provide evidence that PleC activity is responsible for the asymmetric distribution of CpaE and itself to only one of the two cell poles. Thus, a polar signal transduction protein controls its own asymmetric location as well as that of a factor assembling a polar organelle.