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Undesired immune responses against protein therapeutics may adversely affect the pharmacokinetics, efficacy, and safety of the product. The presence of anti-drug-antibodies (ADA) has been the key determinant of immunogenic responses. Here we describe the use of a capillary electrophoresis platform for the identification of ADAs against several experimental camelid VHH biologics (Nanobodies®). Hereafter, we refer to this assay as WESADA. We modified the Wes platform by ProteinSimple to screen serum samples for ADA against covalently linked multi-modular Nanobodies and compared it to standard ADA methodologies. We were able to identify ADA positive samples and determine which individual VHH module in a multivalent Nanobody construct stimulated the predominant ADA response. WESADA requires denaturation of the experimental immobilized drug, which could affect recognition of the immunogenic epitope and alter ADA signal. To address this issue, we demonstrated that signal can be immunodepleted by pre-incubation of serum samples with native Nanobody. This capillary electrophoresis based approach allows for rapid analysis without the need for individually tailored assay optimization or reagent labeling, while consuming small amounts of sample and drug. It also allows for the simultaneous ADA analysis of multiple targets of different molecular size in the same experimental sample. WESADA is not intended to replace traditional ADA assay formats, but it facilitates the expedient immunogenic assessment of a large number of experimental drug candidates in the early developmental space. • A capillary electrophoresis-based method for detecting anti-drug antibodies (ADA). • Murine ADA against bispecific anti-mPD1/anti-mLAG3 Nanobodies. • Rapid ADA analysis with minimal sample volume and reagent requirements. • Novel ADA assay without the need for drug-specific assay optimization. [ABSTRACT FROM AUTHOR]

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo. [ABSTRACT FROM AUTHOR]

IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications. [ABSTRACT FROM AUTHOR]

Abstract: Drug design efforts in the emerging 2-aminothiazole-4-carboxamide class of CHK1 inhibitors have uncovered specific combinations of key substructures within the molecule; resulting in significant improvements in cell-based activity while retaining a greater than one hundred-fold selectivity against CDK2. The X-ray crystal structure of a complex between compound 39 and the CHK1 protein detailing a ‘U-shaped’ topology and key interactions with the protein surface at the ATP site is also reported. [Copyright &y& Elsevier]

Abstract: Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors. [Copyright &y& Elsevier]

Abstract: The synthesis and hit-to-lead SAR development of a pyrazolo[1,5-a]pyrimidine hit 4 is described leading to a series of potent, selective CHK1 inhibitors such as compound 17r. In the Letter, the further utility of the pyrazolo[1,5-a]pyrimidine template for the development of potent, selective kinase inhibitors is detailed. [Copyright &y& Elsevier]

Abstract: A novel series of CHK1 inhibitors based on thienopyridine template has been designed and synthesized. These inhibitors maintain critical hydrogen bonding with the hinge and conserved water in the ATP binding site. Several compounds show single digit nanomolar CHK1 activities. Compound 70 shows excellent enzymatic activity of 1nM. [ABSTRACT FROM AUTHOR]

Abstract: A series of four bicyclic cores were prepared and evaluated as cyclin-dependent kinase-2 (CDK2) inhibitors. From the in-vitro and cell-based analysis, the pyrazolo[1,5-a]pyrimidine core (represented by 9) emerged as the superior core for further elaboration in the identification of novel CDK2 inhibitors. [Copyright &y& Elsevier]

Abstract: In the retina, integrins in the β1 family have been shown to be important in many phases of neuronal development, particularly neuroblast migration and axon outgrowth. However, the functions of specific integrin heterodimers are not well defined. In this study, we investigated the functions of β1 integrins in developing chicken retina by expression of a dominant-negative β1A construct using a replication-competent retrovirus. Inhibition of integrins using this approach resulted in alteration of cell morphology and increased apoptosis, but did not preclude migration and axon elongation. In an attempt to identify which specific β1 heterodimer was important, expression and function of the α4β1 heterodimer were also investigated. At early developmental stages, α4 protein and mRNA were detected in undifferentiated neuroblasts throughout the retina. At later stages, expression was confined to retinal ganglion cells (RGCs) and amacrine cells. A small molecule antagonist of α4 integrins was shown to inhibit neurite outgrowth on recombinant soluble vascular cell adhesion molecule-1 (VCAM-1), a known ligand of α4β1. Introduction of α4 antagonist in vivo gave rise to increased apoptosis and led to a thinning of the retina and reduced numbers of retinal ganglion cells (RGCs). We conclude that the integrin α4β1 is important for survival of developing retinal neurons, including RGCs. [Copyright &y& Elsevier]

Peggy Sue is a capillary-based western/immunoassay platform that can separate and characterize proteins by size or charge. A quick and automated immunogenicity assay was developed on Peggy Sue based on charge separation and compared with a traditional bridging method using preclinical samples from non-human primate studies. The results generated with the Peggy Sue assay were comparable to those of the bridging assays. The Peggy Sue platform has several advantages, including time efficiency, low sample consumption, and easy automation. The platform is especially ideal for further characterization of anti-drug antibody (ADA) specificity against complex biologics such as bispecific or multi-specific biotherapeutics as it is easy to conduct domain specificity assessment of observed ADA responses. Our evaluation suggests that the Peggy Sue platform is a promising tool for preclinical ADA analysis. [ABSTRACT FROM AUTHOR]