Neurofilament light chain (NfL) is a pivotal biomarker for neuroaxonal damage. However, its quantification in volume-limited matrices, such as mouse aqueous humor (AH) (2 to 6 µL per eye), has historically required pooling samples from multiple animals due to the low sensitivity and high sample volume requirements of standard immunoassays. Such pooling increases animal requirements and precludes individual-level data analysis. To overcome these limitations, we developed and evaluated a NfL HomeBrew (HB) assay using the NUcleic acid-Linked Immuno-Sandwich Assay (NULISA) technology. NULISA offers superior analytical sensitivity and requires less than 10 µL volume. Our results demonstrate that the NfL HB NULISA performs comparably to the Simoa reference assay and provides superior sensitivity to the Ella platform. Crucially, the assay reliably quantified NfL in as little as 2 µL of individual mouse AH, with 90

BackgroundTechnologies such as ELISA, MSD, and Gyrolab have been employed for quantifying protein therapeutics in clinical trials. However, these technologies have limitations with dynamic range often requiring multiple dilution steps, introducing potential errors and variability.Results/methodologyA pharmacokinetics assay was successfully developed on the NUcleic acid Linked Immuno-Sandwich Assay (NULISA) platform with a concentration dynamic range exceeding 6 logs. This enabled assessment of all clinical samples across different concentrations with a single dilution, yielding results with good correlation to ELISA and Gyrolab.ConclusionsNULISA technology offers high sensitivity, full automation, and a wide dynamic range, streamlining assay development and optimization, simplifying sample analysis, minimizing errors, and increasing throughput.

In preclinical protein therapeutic development studies, the emergence of anti-drug antibodies (ADA) can potentially impact drug pharmacokinetics and safety. While immunogenicity assessment is not mandatory in preclinical studies, banking samples can be valuable for interpreting unexpected pharmacological responses. Immunoassays that use generic reagents across different drug molecules can simplify ADA assessment and expedite sample evaluations. This work showcases the ability of the Gyrolab automated immunoassay platform to detect and quantify both drug-free and drug-bound (total) ADAs to monoclonal antibody (mAb) therapeutics in cynomolgus monkey preclinical studies. Compared to the previously reported total ADA ELISA, the Gyrolab assay exhibited a wider signal dynamic range and increased drug tolerance. Similar sensitivity, dynamic range and cut point factors were observed for four therapeutic mAbs of different isotypes using the Gyrolab assay. Here we present a comparison of ADA assays using bridging ELISA, total ADA ELISA and total ADA Gyrolab formats in a cynomolgus monkey study where the subjects were treated with a single dose of a mAb therapeutic. We demonstrate that the total ADA assays detected host ADA responses at earlier time points compared to the bridging ELISA. The Gyrolab assay has the best correlation between signal-to-noise (S/N) and titer over a wide ADA concentration range, highlighting the utility of Gyrolab in S/N reporting of ADA response to eliminate the need for secondary titer assays. Collectively, our results demonstrate that the generic ADA Gyrolab assay minimizes the necessity for extensive assay development and optimization for therapeutic mAbs, streamlining preclinical immunogenicity assessment to enable interpretation of pharmacological data.

The measurement of therapeutic drug concentrations is used to assess drug exposure and the relationship between therapeutic pharmacokinetics (PK) and pharmacodynamics (PD), which help determine the optimal dose for patients. Ligand binding assays (LBAs) are often the method of choice for evaluation of drug concentration and use either the therapeutic target protein or antibodies to the therapeutic as capture and/or detection reagents. Due to the bivalency of antibody therapeutics, heterogeneous states of the drug/target complex can exist in the presence of soluble targets which can complicate measurement of unbound drug. In the case of bispecific antibodies, measurement of drug can be even more complicated and depend upon the levels of both targets to each arm. Measuring the total drug allows for PKPD modeling prediction of human dose projections in addition to overcoming challenges associated with measuring free drug for bispecific antibodies. Here, we present a study in which a sandwich ELISA format was used to measure total anti-KLK5/KLK7 antibody concentrations. This assay utilized a non-blocking anti-idiotype (ID) antibody to one arm of the antibody for capture and an antibody to target bound to the other arm of the antibody for detection. Our qualified assay showed acceptable precision, accuracy, dilutional linearity, and reproducibility and enabled detection of a total bispecific antibody at high levels of two targets. To confirm that our assay was detecting total drug, a subset of samples was evaluated in a generic total LC–MS/MS assay.

RO7449135, an anti-kallikrein (KLK)5/KLK7 bispecific antibody, is in development as a potential therapy against Netherton's syndrome (NS). In cynomolgus monkey studies, RO7449135 bound to KLK5 and KLK7, causing considerable accumulation of total KLKs, but with non-dose-proportional increase. To understand the complex PKPD, a population model with covariate analysis was developed accounting for target binding in skin and migration of bound targets from skin to blood. The covariate analysis suggested the animal batch as the categorical covariate impacting the different KLK5 synthesis rates between the repeat-dose study and single-dose study, and the dose as continuous covariate impacting the internalization rate of the binary and ternary complexes containing KLK7. To comprehend the mechanism underlying, we hypothesized that inhibition of KLK5 by RO7449135 prevented its cleavage of the pro-enzyme of KLK7 (pro-KLK7) and altered the proportion between pro-KLK7 and KLK7. Besides the pro-KLK7, RO7449135 can interact with other proteins like LEKTI through KLK7 connection in a dose-dependent manner. The different high-order complexes formed by RO7449135 interacting with pro-KLK7 or LEKTI-like proteins can be subject to faster internalization rate. Accounting for the dose and animal batch as covariates, the model-predicted free target suppression is well aligned with the visual target engagement check. The population PKPD model with covariate analysis provides the scientific input for the complex PKPD analysis, successfully predicts the target suppression in cynomolgus monkeys, and thereby can be used for the human dose projection of RO7449135.

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure-function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure-function relationship of terminal galactose to complement activation in mAb therapeutics.

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit patients' T cells to kill cancer cells. There are many TDBs in clinical trials, demonstrating their widely recognized therapeutic potential. However, their complex, multi-step mechanism of action (MoA), which includes bispecific antigen binding, T-cell activation, and target-cell killing, presents unique challenges for biological characterization and potency assay selection. Here, we describe the development of a single reporter-gene potency assay for a TDB (TDB1) that is MoA reflective and sensitive to binding of both antigens. Our reporter-gene assay measures T-cell activation using Jurkat cells engineered to express luciferase under the control of an NFkB response element. The potencies of select samples were measured both by this assay and by a flow-cytometry-based cell-killing assay using human lymphocytes as effector cells. Correlating the two sets of potency results clearly establishes our reporter-gene assay as MoA reflective. Furthermore, correlating potencies for the same panel of samples against binding data measured by binding assays for each individual arm demonstrates that the reporter-gene potency assay reflects dual-antigen binding and can detect changes in affinity for either arm. This work demonstrates that one reporter-gene assay can be used to measure the potency of TDB1 while capturing key aspects of its MoA, thus serving as a useful case study of selection and justification of reporter-gene potency assays for TDBs. Furthermore, our strategy of correlating reporter-gene potency, target-cell killing, and antigen binding for each individual arm serves as a useful example of a thorough, holistic approach to biological characterization for TDBs that can be applied to other bispecific molecules.

Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work. FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5-20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc.

Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand-bearing osteoblastic or stromal cells inhibits wild type but not O-fucosylglycan-deficient HSPC cycling, independent of RBP-JK-mediated canonical Notch signaling. Furthermore, Notch-ligand neutralizing antibodies induce RBP-JK-independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor–ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O-fucosylglycans on Notch. Stem Cells 2015;33:2280–2293

BACKGROUND: Notch has long been recognized as an important molecule that regulates stem cell self-renewal and differentiation. Despite that Notch is required for the embryonic hematopoietic stem cell (HSC) generation, and that Notch is expressed in the adult HSC, the role of Notch pathway in adult HSC remains unclear. Recently it has been shown that Notch activation and the ability of interacting with Notch ligand Jagged1 is a signature of human primitive HSC and supports HSC regenerative potential. Here we study the physiological significance of Notch in the adult HSC population focusing on how Notch-ligand engagement regulates HSC quiescence and niche retention.METHODS: To better understand the role of Notch in adult HSC homeostasis, we examined HSC frequency, quiescence maintenance, niche occupancy, and HSC mobilization in mice with either conditional lack of RBP-JK, which mediates the canonical Notch signaling activity, or in mice with conditional lack of Pofut1 that catalyzes O-fucosylation of Notch EGF-like repeats and the generation of O-fucose glycans important for the binding of Notch ligands. We also tested Notch ligand neutralizing antibodies and Notch1 and Notch2 inhibitory antibodies to examine the effect of blocking Notch receptor-ligand engagement or the block of Notch signaling activation on HSC homeostasis.RESULTS: We report here that Pofut1-deficient hematopoietic stem and progenitor cells (HSPCs) display enhanced cell cycling and proliferation cell autonomously. These changes are accompanied by G-CSF-independent increased HSPC egress from the marrow to the periphery and other hematopoietic organs, and their enhanced sensitivity to mobilizing stimuli of G-CSF plus the CXCR4 antagonist, AMD3100. This phenotype is caused by reduced adhesion of Pofut1-deficient HSPC to Notch ligand-expressing stromal cells and the osteoblastic lineage cells. Adhesion to these cells by wild type but not Pofut1-deficient HSPCs can be blocked by recombinant Notch ligand Dll1 or Dll4. In addition, adhesion to these cells inhibits wild type but not Pofut1-deficient HSPC cycling, independent of RBP-Jk-mediated Notch signaling. Further, Pofut1-deficient HSPCs exhibit normal expression of key adhesion molecules and normal SDF-1-mediated chemotaxis, but show scattered and distal occupancy in the endosteal or osteoblastic niche. In support of roles for Notch-ligand engagement in facilitating HSPC niche retention, we show that mice receiving 4 doses of neutralizing antibodies to the Notch ligand Dll4 or Jagged1, but not Dll1, display ~2-3-fold increased HSC and progenitor egress when compared to mice receiving isotype control antibody, and further display 60% increased HSC mobilization when compared to mice receiving control antibody and treated similarly with G-CSF and AMD3100. Dll4 blockade also increases the sinusoidal endothelial cell population and HSPC cell cycling. In comparison, only a mild HSPC proliferation and egress is found in RBP-JK-deficient mice, or in mice receiving Notch2 inhibitory antibody. However, Notch2 blockade but not Notch1 blockade induces unique features of HSC and myeloid progenitor mobilization responding to G-CSF plus AMD3100.CONCLUSIONS: Based on these findings, we conclude that HSPC quiescence and retention in the marrow niche is facilitated by the interaction between Notch-expressing HSPCs and Jagged1- or Dll4-expressing niche cells, and is likely also contributed by Notch signaling activation. In addition, Notch receptor-ligand engagement in this process is strengthened by O-fucose modification of Notch receptors. Finally, the observations from our studies may provide a therapeutic indication. Inadequate mobilization in HSPC transplantation remains a clinical problem. Our findings that targeting Notch receptor-ligand interaction and/or inhibiting Notch2 activation increase HSPC emigration suggests a novel approach for enhancing mobilization of stem and progenitor cells for those patients who respond poorly to current mobilizing regimes.Disclosures Shim: Genentech: Employment, Equity Ownership. Yan: Genentech: Employment, Equity Ownership. Lowe: Genentech: Employment, Equity Ownership. Siebel: Genentech: Employment, Equity Ownership.

Signaling from the Notch receptor family is important in development, and its role in disease has assigned the Notch signaling pathway as a therapeutic target. Mammalian Notch receptors are single‐pass transmembrane glycoproteins containing 29–36 EGF‐like repeats. Notch receptors on “receiving” cells signal when they interact with Notch ligands expressed on “sending” cells. Glycosylation of some Notch EGF‐like repeats modulates Notch‐dependent signaling. EGF repeat glycosylation includes constitutive fucosylation of some EGF repeat‐localized serine/threonine residues, and elongation of fucose‐initiated glycans regulated by the Fringe family of glycosyltransferases.To understand how glycosylation controls Notch signaling, we sought to determine how O‐fucosylated glycans modulate Notch receptor‐ligand interactions, using (1) surface plasmon resonance, (2) cell‐based binding assays, and (3) cell‐based Notch signaling assays. We find that lunatic Fringe modification of Notch1 receptors enhances Dll1 and Dll4 binding affinities and signaling, while it reduces Jag1‐mediated binding affinity and signaling. Fringe modification of Notch1 does not significantly alter Jag2‐mediated binding affinity and signaling. We also find that Dll1 binding and signaling is enhanced by lunatic Fringe primarily when Notch1 expression is low, and this enhancement is blunted when Notch1 expression is high.

Notch1 is modified by O‐fucosylation catalyzed by Protein O‐fucosyltransferase 1 (Pofut1). The Fringe family of glycosyltransferases extend Notch‐borne O‐fucose moieties, and modulate Notch1 signaling by altering the sensitivity of Notch receptors to Notch ligands. To address the physiologic function Notch O‐fucosylation in myelopoiesis, we characterized myelopoiesis in mice carrying a deficiency in fucosylation (the FX(−/−) mouse), or with an inducible deficiency of Pofut1. In either strain, loss of Notch fucosylation yields a myeloid hyperplasia phenotype. Restoration of Notch1 signaling suppresses myeloid hyperplasia. Loss of fucosylation in FX(−/−) mice or in Pofut1‐deficient mice is associated with diminished cell surface expression of Notch family members, loss of binding of Notch ligands to the fucosylation‐deficient cells in the mice, and suppressed activation of Notch target genes. Surface plasmon resonance experiments indicate that Fringe‐mediated elongation of O‐fucosylated Notch modulates binding of Notch ligands to Notch1, corresponding to Fringe‐dependent modulation of Notch1‐dependent signal transduction. These observations indicate that Pofut1‐dependent Notch fucosylation is required for myelopoietic homeostasis via proper (i) cell surface localization of Notch1, (ii) binding of Notch ligands to Notch1, and (iii) Fringe‐dependent modulation of Notch1 signaling.

Notch receptors are cell surface molecules essential for cell fate determination. Notch signaling is subject to tight regulation at multiple levels, including the posttranslational modification of Notch receptors by O-linked fucosylation, a reaction that is catalyzed by protein O-fucosyltransferase-1 (Pofut1). Our previous studies identified a myeloproliferative phenotype in mice conditionally deficient in cellular fucosylation that is attributable to a loss of Notch-dependent suppression of myelopoiesis. Here, we report that hematopoietic stem cells deficient in cellular fucosylation display decreased frequency and defective repopulating ability as well as decreased lymphoid but increased myeloid developmental potential. This phenotype may be attributed to suppressed Notch ligand binding and reduced downstream signaling of Notch activity in hematopoietic stem cells. Consistent with this finding, we further demonstrate that mouse embryonic stem cells deficient in Notch1 (Notch1(-/-)) or Pofut1 (Pofut1(-/-)) fail to generate T lymphocytes but differentiate into myeloid cells while coculturing with Notch ligand-expressing bone marrow stromal cells in vitro. Moreover, in vivo hematopoietic reconstitution of CD34(+) progenitor cells derived from either Notch1(-/-) or Pofut1(-/-) embryonic stem cells show enhanced granulopoiesis with depressed lymphoid lineage development. Together, these results indicate that Notch signaling maintains hematopoietic lineage homeostasis by promoting lymphoid development and suppressing overt myelopoiesis, in part through processes controlled by O-linked fucosylation of Notch receptors.

Cell-cell contact-dependent mechanisms that modulate proliferation and/or differentiation in the context of hematopoiesis include mechanisms characteristic of the interactions between members of the Notch family of signal transduction molecules and their ligands. Whereas Notch family members and their ligands clearly modulate T lymphopoietic decisions, evidence for their participation in modulating myelopoiesis is much less clear, and roles for posttranslational control of Notch-dependent signal transduction in myelopoiesis are unexplored. We report here that a myeloproliferative phenotype in FX(-/-) mice, which are conditionally deficient in cellular fucosylation, is consequent to loss of Notch-dependent signal transduction on myeloid progenitor cells. In the context of a wild-type fucosylation phenotype, we find that the Notch ligands suppress myeloid differentiation of progenitor cells and enhance expression of Notch target genes. By contrast, fucosylation-deficient myeloid progenitors are insensitive to the suppressive effects of Notch ligands on myelopoiesis, do not transcribe Notch1 target genes when cocultured with Notch ligands, and have lost the wild-type Notch ligand-binding phenotype. Considered together, these observations indicate that Notch-dependent signaling controls myelopoiesis in vivo and in vitro and identifies a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in myeloid progenitors.