Antibody Drug Conjugate (ADC) Drugs Kill Target Cells

• What is an Antibody Drug Conjugate (ADC)?
• What potency assays are needed for an ADC?
• How can Marin Biologic Laboratories help you with bioassays? 

What is an Antibody Drug Conjugate?

Antibodies are very specific for a target.  In an ADC, a toxic substance is conjugated to an antibody to kill a target cell after binding.  Examples of toxic conjugates are Calicheamicin, a microbial inhibitor of DNA synthesis, Auristatin, a synthetic inhibitor of tubulins, and the anti-neoplastic Exatecan.  An example of an ADC is shown below.

How Does an Antibody Drug Conjugate Kill Cells?

ADCs combine the targeting properties of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs, designed to discriminate between healthy and diseased tissue.  The antibody binds to the target cell with exquisite specificity and delivers the toxic drug locally. Usually, the drug is internalized via antibody receptors and the cytotoxin does its damage intracellularly.

Some ADCs are designed to kill regulatory T lymphocytes that protect malignancies from immune attack.

The ADC market size is expected to reach US $34 billion by 2028.

ADC Potency Assays

1.Binding.  In choosing a candidate ADC, the first step is high affinity binding and sufficient number of receptors per cell. Cell binding assays are employed to assess the interaction between a drug candidate and its target receptors on cells. These assays help determine the drug’s affinity and specificity for the target, aiding in the selection of lead compounds and optimizing their binding characteristics during drug development. Ensuring consistent and reproducible results in GMP-compliant cell binding assays is essential for quality control and batch-to-batch consistency.

Assay development includes the unconjugated antibody. Additionally, various conjugates are tested for binding to target cells. Under GMP manufacturing, both substances are tested as lot release criteria. The methods used are ELISA and other affinity methods.

Case Study from Marin Biologic Laboratories:

Target cancer cells were fixed with paraformaldehyde and incubated with a reference standard and a separate test lot of an ADC in a 96 well plate. All conditions were tested in triplicate.  Bound ADC is detected using a fluorescent antibody to the ADC.  The graph below shows very close agreement of triplicate values of Relative Fluorescence Units (RFU) and close agreement of the reference standard (RS) to the test sample, EC₅₀ = 0.302 ng/mL and 0.313 ng/mL, respectively.

2.Toxicity. Cell toxicity assays measure the potency of a drug to kill cells. They are also used to assess the potential harmful effects of a drug on cells, helping identify toxic compounds early in the development process. GMP-compliantcell toxicity assays ensure that drug formulations are safe for clinical trials and eventual commercialization.

For the toxicity assay, the ADC is incubated at increasing concentrations with the target cell for a set period of time. After incubation, toxicity is measured by a variety of techniques.

Case Study from Marin Biologic Laboratories:

Target cancer cells were incubated with a reference standard (RS) and a different lot of an ADC (Test) for four days. All conditions were tested in triplicate. Viability was assessed by luminescence (Lum) using the CTG reagent (Promega) that detects intracellular ATP of live cells.  The graph below shows the large dynamic range and excellent precision of results and agreement of the test sample with the reference standard, EC₅₀ = 4.61 ng/mL and 4.51 ng/mL, respectively.

Improved Design ADCs

Degrader Antibody Conjugates (DAC) targeted protein degradation has emerged as a new paradigm to manipulate cellular proteolysis.  Proteolysis-targeting chimeras (PROTACs) are bifunctional small molecules that recruit an enzyme to a target protein of interest, promoting its degradation.   Antibody-based PROTACs (AbTACs) incorporate this principle with unique antibody directing activity.

An example is a DAC that degrades an immune cell surface checkpoint protein programmed death-ligand 1 (PD-L1). Degradation of this protein that is often over-expressed on cancer cells should lead to better destruction by immune cells. Upon contact with an appropriate cell, the PD-L1 intracellular region is brought into contact with a cell surface enzyme and is degraded. For more information, click the link below.

Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1

Another example is an ADC that degrades a protein in HER2 positive breast cancers that is required for tumor growth. For more information, click the link below.

HER2-Dependent BRD4 Protein Degradation by Antibody−PROTAC Conjugates

How can Marin Biologic Laboratories help?

Marin Biologic Laboratories provides solutions in protein/gene analytical and biological assays and other services for our many clients, with or without GLP/GMP conditions. Marin Biologic develops, validates, and performs a variety of assays for

• GMP potency assay for Lot Release, GLP assays
• ELISA, PK and PD studies, Immunogenicity (Anti-Drug Antibodies), Neutralizing Antibodies
• Humoral and Cellular Immunity Assays. 
• Cell-based assays, proliferation and toxicity assays
• Molecular, Biochemical and Biology services, Protein Production, Purification, Enzyme Assays, Cloning, Transfections
• Radioactivity procedures and analysis. 

Marin Biologic Laboratories is a contract laboratory for pharmaceutical and biotech industries. We have been operating for over 25 years and we take pride in our science as well as the quality of our communication. We specialize in development and validation of cell-based assays and immunoassays as well as enzyme assays, protein and RNA expression, transfections, protein purification, and radioactivity methods.  We are FDA inspected.  We would like to help meet your requirements in a timely and cost effective way.