Lumit: A Homogeneous Bioluminescent Immunoassay for Detecting Diverse Analytes and Intracellular Protein Targets.

Traditional immunoassays to detect secreted or intracellular proteins can be tedious, require multiple washing steps, and are not easily adaptable to a high-throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. This bioluminescent immunoassay does not require washes or liquid transfers and takes less than 2 h to complete in a homogeneous "Add and Read" format. In this chapter, we describe step-by-step protocols to create Lumit immunoassays for the detection of (1) secreted cytokines from cells, (2) phosphorylation levels of a specific signaling pathway node protein, and (3) a biochemical protein-protein interaction between a viral surface protein and its human receptor.

A homogeneous bioluminescent immunoassay for parallel characterization of binding between a panel of antibodies and a family of Fcγ receptors.

Fc engineering efforts are increasingly being employed to modulate interaction of antibodies with variety of Fc receptors in an effort to improve the efficacy and safety of the therapeutic antibodies. Among the various Fc receptors, Fc gamma receptors (FcγRs) present on variety of immune cells are especially relevant since they can activate multiple effector functions including antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Depending on the desired mechanism of action (MOA) of the antibody, interactions between Fc domain of the antibody and FcγR (denoted as Fc/FcγR) may need to be enhanced or abolished. Therefore, during the antibody discovery process, biochemical methods are routinely used to measure the affinities of Fc/FcγR interactions. To enable such screening, we developed a plate based, simple to use, homogeneous immunoassays for six FcγRs by leveraging a luminescent protein complementation technology (NanoBiT). An added advantage of the NanoBiT immunoassays is their solution-based format, which minimizes well known surface related artifacts associated with traditional biosensor platforms (e.g., surface plasmon resonance and biolayer interferometry). With NanoBiT FcγRs assays, we demonstrate that assays are specific, report IgG subclass specific affinities and detect modulation in Fc/FcγR interactions in response to the changes in the Fc domain. We subsequently screen a panel of therapeutic antibodies including seven monoclonal antibodies (mAbs) and four polyclonal intravenous immunoglobulin (IVIg) products and highlight the advantages of parallel screening method for developing new antibody therapies.

Bioluminescent Bridging Immunoassay for Anti-Drug Antibody (ADA) Detection.

Any immune reaction to therapeutic antibodies will impact the drug efficacy and can have serious consequences for patient safety. Therefore, detection and reporting of anti-drug antibodies (ADA) during clinical trials is required by regulatory agencies during drug approval process. We have developed a bioluminescent bridging immunoassay for ADA detection, which uses an extremely bright NanoLuc enzyme as a reporter. The assay is sensitive with a wide dynamic range and meets the FDA drug tolerance guideline of detecting 100 ng/ml of ADA in the presence of 500-fold excess of free drug. We describe detailed protocols for development of ADA assays using therapeutic Trastuzumab as a model drug and an anti-Trastuzumab antibody as an example of immune response.

Deciphering the Interaction between Neonatal Fc Receptor and Antibodies Using a Homogeneous Bioluminescent Immunoassay.

Long half-life of therapeutic Abs and Fc fusion proteins is crucial to their efficacy and is, in part, regulated by their interaction with neonatal Fc receptor (FcRn). However, the current methods (e.g., surface plasmon resonance and biolayer interferometry) for measurement of interaction between IgG and FcRn (IgG/FcRn) require either FcRn or IgG to be immobilized on the surface, which is known to introduce experimental artifacts and have led to conflicting data. To study IgG/FcRn interactions in solution, without a need for surface immobilization, we developed a novel (to our knowledge), solution-based homogeneous binding immunoassay based on NanoBiT luminescent protein complementation technology. We optimized the assay (NanoBiT FcRn assay) for human FcRn, mouse FcRn, rat FcRn, and cynomolgus FcRn and used them to determine the binding affinities of a panel of eight Abs. Assays could successfully capture the modulation in IgG/FcRn binding based on changes in Fc fragment of the Abs. We also looked at the individual contribution of Fc and F(ab)2 on the IgG/FcRn interaction and found that Fc is the main driver for the interaction at pH 6. Our work highlights the importance of using orthogonal methods to validate affinity data generated using biosensor platforms. Moreover, the simple add-and-read format of the NanoBiT FcRn assay is amenable for high-throughput screening during early Ab discovery phase.

Development of NanoLuc bridging immunoassay for detection of anti-drug antibodies.

Anti-drug antibodies (ADAs) are generated in-vivo as an immune response to therapeutic antibody drugs and can significantly affect the efficacy and safety of the drugs. Hence, detection of ADAs is recommended by regulatory agencies during drug development process. A widely accepted method for measuring ADAs is "bridging" immunoassay and is frequently performed using enzyme-linked immunosorbent assay (ELISA) or electrochemiluminescence (ECL) platform developed by Meso Scale Discovery (MSD). ELISA is preferable due to widely available reagents and instruments and broad familiarity with the technology; however, MSD platform has gained wide acceptability due to a simpler workflow, higher sensitivity, and a broad dynamic range but requires proprietary reagents and instruments. We describe the development of a new bridging immunoassay where a small (19kDa) but ultra-bright NanoLuc luciferase enzyme is used as an antibody label and signal is luminescence. The method combines the convenience of ELISA format with assay performance similar to that of the MSD platform. Advantages of the NanoLuc bridging immunoassay are highlighted by using Trastuzumab and Cetuximab as model drugs and developing assays for detection of anti-Trastuzumab antibodies (ATA) and anti-Cetuximab antibodies (ACA). During development of the assay several aspects of the method were optimized including: (a) two different approaches for labeling drugs with NanoLuc; (b) sensitivity and dynamic range; and (c) compatibility with the acid dissociation step for improved drug tolerance. Assays showed high sensitivity of at least 1.0ng/mL, dynamic range of greater than four log orders, and drug tolerance of >500.

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads.

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be beneficial for screening a large number of antibody samples during early monoclonal development phase.

On-bead antibody-small molecule conjugation using high-capacity magnetic beads.

Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Methods for the purification of HQ-tagged proteins.

The HQ (H = histidine, Q = glutamine) tag is a small fusion tag that can be isolated using immobilized metal affinity columns. HQ-tagged proteins can be expressed and purified from bacterial cells under native and denaturing conditions, mammalian cells, insect cells, wheat germ and rabbit reticulocyte. Furthermore, HQ-tagged proteins can be purified using magnetic or non-magnetic resins in multiple formats from small to large-scale and manual or automated. In this chapter, we have described various protocols for the purification of HQ-tagged proteins.