Antibody Drug Development
Reagents & Assays to Support Drug Discovery
Protein- and antibody-based therapeutics have revolutionized medicine. The first protein-based therapeutic, insulin, was administered to diabetic patients to regulate blood sugar in 1922. Since then, roughly 60 more proteins have been approved for clinical use by the United States' Food and Drug Administration (FDA). Antibody-based therapeutics have become increasingly popular since the first one was approved by the FDA in 1986 (Figure 1), with the 100th therapeutic antibody approved in 2021. Protein- and antibody-based therapeutics help treat a plethora of human diseases, such as cancer, blood disorders, rheumatoid arthritis, psoriasis, multiple sclerosis, high cholesterol, and systemic lupus erythematosus. They are also used to manage weight gain, growth, and pregnancies.
RayBiotech has a comprehensive catalog of products and services to advance drug discovery, from off-the-shelf arrays to screen for drug candidates to custom assays and services. Our comprehensive analytical services ensure compliance with CLIA, good clinical laboratory practices (GCLP), good laboratory practice (GLP), and current good manufacturing practice (cGMP) standards as well as current regulatory guidance documents based on industry requirements.
Figure 1. Number of DAs approved by the FDA (1986 – 2020). Data from the Antibody Society and Lu, RM., Hwang, YC., Liu, IJ. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020).
Drug Antibody (DA) Detection
A drug antibody (DA) is an antibody that elicits the destruction of disease-specific cells directly or indirectly. Also known as a therapeutic antibody, the primary mechanism of action of DAs is the specific and stable interaction with a unique target molecule called an "antigen" on the cells-of-interest, which either induces cell apoptosis or recruits immune cells to destroy the target cells. As such, DAs are a type of immunotherapy that cause fewer adverse effects than systemic drugs (e.g., chemotherapy) that now account for a fifth of all new drug approvals.
Modifications can further enhance the therapeutic efficacy of DAs by increasing delivery specificity, improving binding affinity, or resulting in a drug-conjugate additive effect. For example, a DA may act as a delivery system that transports its radionuclide conjugate directly to tumor cells, thus sparing healthy tissues from unnecessary radiation.
DA assays characterize DA binding, identify candidate DAs, or monitor DA lot-to-lot variability. An example of one such assay, the bridging ELISA, is depicted in Figure 2. Epitope mapping of DAs can provide crucial information about their therapeutic functions. Figure 3 shows how custom-designed peptide arrays can be used to identify key binding sites on the drug target which are recognized by the DA.
Anti-Drug Antibody (ADA) Detection
Anti-drug antibodies (ADAs) can be produced by the body after repeated treatment with DAs. ADAs decrease DA efficacy by reducing its concentration or interfering with its binding to the target molecule. ADAs that interfere with DA binding are known as neutralizing antibodies. Therefore, the identification of DAs with low immunogenicity - or DAs that have a minimal propensity to generate an immune response - is essential in drug discovery.
ADAs are first screened with assays that detect binding of endogenous ADAs to DAs. An ADA assay using flow cytometry is shown in Figure 4. Further characterization of ADAs may include titration, neutralization, epitope mapping, isotyping, and cross-reactivity assays. The ADA titer and neutralizing activity may provide important insight into the pharmacokinetics, pharmacodynamics, safety, and therapeutic efficacy of DAs. Drug design can be improved with epitope mapping, which identifies the DAs' immunogenic regions (Figure 3). Isotyping determines the specific immune response to DAs. Finally, cross-reactivity assays that determine which endogenous and exogenous proteins are targeted by the DAs can guide patient care.
Protein-based drugs are usually synthetic versions of proteins that are produced naturally by the body. They modify cellular responses by either acting directly with the targets-of-interest (see also Receptor/Ligand Binding), recruiting other molecules to the site, or improving the function of nearby cells. For example, interleukin 2 (IL-2) is used in kidney and skin cancer treatment to interfere with cancer growth, recruit immune cells, and stimulate the production of immune cells that can help destroy the cancer cells. Lactase, which is used by individuals who are lactose intolerant to break down lactose, is another example of a protein-based drug. Like DAs, drug proteins can stimulate the production of anti-drug protein antibodies that can decrease therapeutic efficacy.
Assays for protein therapeutics are used to detect drug proteins, drug protein interactions, and anti-drug protein antibodies.
Receptors are transmembrane proteins on the cell surface that bind to specific ligands called "agonists." This interaction activates the receptor, setting off a series of downstream signaling events that result in a specific cellular response (e.g., proliferation). Such responses, however, can be blocked if an "antagonist" binds to the receptor instead without activating it. Pathogens, such as viral particles, can bind to receptors to facilitate infection or other undesired effects.
Identifying receptor antagonists or other molecules that inhibit unwanted receptor activation is a focus of drug discovery efforts. Binding assays like the one shown in Figure 5 are used to screen for antibodies, peptides, proteins, and small molecules that block ligand-receptor interactions.
A drug may cross-react with non-target proteins, which can result in adverse side effects or expand its indications. The characterization of drug interactions and its downstream consequences is paramount in drug development.
RayBiotech's antibody arrays enable the detection of multiple proteins simultaneously. As many as 1200 human proteins or 640 mouse proteins can be quantified. Even higher-density arrays for semi-quantitative data are available with our L-Series arrays, analyzing as many as 4000 human, 1500 rat, or 1308 mouse proteins at the same time.
Biomarkers and enriched signaling pathways following drug treatment can be identified with RayBiotech's biostatistics and bioinformatics services. Data can then be validated using a custom sandwich-based antibody array that uses an antibody pair for high specificity. A capillary-based alternative to western blotting, auto-western, can also be employed for biomarker validation.
Proteins and antibodies are conjugated to molecules to enable detection or binding. These molecules may include other proteins, fluorophores, biotin, and beads. If you need custom conjugation services, please e-mail us at [email protected].
This EC50 determination for antibody employs an indirect ELISA method. In this assay, standard 96-well plates (12 strips with 8 wells/strip) are coated with a specific protein, which combines with the corresponding antibody present in a sample, which is diluted in a series of dilution fold for EC50 determination. The wells are washed, and biotinylated detection antibody such as anti-human IgG antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells, and color develops in proportion to the amount of the specific protein antibody bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. The EC50 concentration can be calculated by plot the series dilution antibody or a sample as 4-PL (4 parametric logistic) standard curve, with standard concentration on the x-axis and absorbance on the y-axis.
Figure 2. Schematic showing how the DA 'bridging' ELISA works. The intensity of color development in the wells is proportional to the amount of DA bound.
Figure 3. Peptide-based epitope mapping. (A) Schematic showing that an antibody binds to its specific epitope on an array with tiled peptides representing the target protein-of-interest. The primary antibody is detected via a biotin-streptavidin-fluorophore complex. (B) A representative fluorescent image of a peptide array obtained with a compatible laser scanner. Each peptide was printed in triplicate. Green spots represent primary antibody binding to the array.
Figure 4. Schematic of a flow cytometry-based ADA assay. Schematic showing a bridging sandwich ELISA-based cytometric bead assay. The ADA is captured in a sandwich immunocomplex and the signal is generated by a phycoerythrin (PE)-streptavidin conjugate.
Figure 5. Schematic showing how a binding assay works. Another format is available where the receptor is in solution and the plate is coated with ligand.