A primary concern in drug development is the binding of drugs to targets they are not supposed to bind to. As a result, off-target effects can occur, which may have severe toxicological consequences in patients. Researchers can use cell microarrays to identify potentially dangerous off-target interactions of new therapeutic molecules to de-risk research programs, increasing patient safety, and improving success rates in regulatory submissions.
Assessing the off-target liability of therapeutic candidates is critical for companies, particularly when filing investigational new drug (IND) data to start in-human clinical trials. To verify that binding of drug candidates occurs in the target tissue only, so-called tissue cross-reactivity (TCR) studies are traditionally conducted.
Drug candidates are incubated with cryosections from a standard panel of human tissues and analyzed by immunohistochemistry (IHC). IHC-based TCR allows therapeutic developers to detect drug-target interactions through enzymatic color changes, which are visualized microscopically.
“IHC-based TCR approaches are excellent tools to determine the ‘where’ – the location of molecular interactions within a histological context,” explained Nick Brown, leader of the UK-based High Peak Client Service Team at Charles River Laboratories.
“But they do not provide much information on the ‘what’ – the identity of the exact protein target(s) the molecule binds to. In addition, some therapeutic molecules are challenging to assess by IHC.”
The where and what of assessing off-target effects
To help address these issues, Retrogenix – which was acquired earlier this year by US-based drug development service provider Charles River Laboratories and now operates as the High Peak site within Charles River – has developed an in vitro off-target screening assay based on cell microarrays.
These microarrays contain cell colonies growing in precisely localized spots, each displaying a defined human protein at the cell surface. When the new molecule, or test article, binds to one of these spots, a distinct signal is detected, enabling identification of the interacting protein.
“Histological (IHC-based) and protein interaction (cell microarray) data ideally complement each other, increasing the reliability, significance, and weight of evidence of off-target study results, particularly at the IND-enabling stage,” Brown said.
Looking at recent internal survey results, 66% of Charles River clients using the Retrogenix Cell Microarray Technology combined both cell microarray and histological data for IND submission.
At the same time, 34% relied on Retrogenix Cell Microarray Technology data only, demonstrating the use of this approach as a standalone method for cross-reactivity assessment.
“We are very proud that 95% of all IND submissions captured in the survey have been approved as of September 2021, with the remaining 5% still in the approvals pipeline,” Brown added.
Detecting off-target effects with cell microarrays
At the core of the Retrogenix Cell Microarray Technology is a proprietary complementary DNA (cDNA) library that contains the genetic information for over 6,200 human plasma membrane and secreted proteins.
Each of these proteins is encoded in a separate piece of DNA that contains all elements needed for protein production, known as expression vectors. These expression vectors are spotted on a defined area on the microarray slides.
When cells are seeded on the microarray slide, they grow over those spots, take up the expression vectors, and start producing the respective human proteins. All proteins, even those that are normally secreted, are designed to be presented at the cell surface, ready to interact with the test article.
“One of the reasons that the Retrogenix Cell Microarray Technology is so powerful is that our cDNA library is the largest, most comprehensive, and highest quality library of its type available globally,” Brown emphasized.
“We have been developing our technology over the last twelve years, screened thousands of samples, and constantly refine, optimize, and expand our library. The next significant update will be released in early December. The library will now include the vast majority of human cell surface protein targets important for Developmental and Reproductive Toxicology (DART) screening, allowing us to extend our offering to assess drug safety for pregnant women.”
Most drug candidates tested are antibodies or antibody-related molecules, though other therapeutic candidates, such as proteins, peptides, whole cells – such as CAR-T cells – and small molecules can be profiled.
Notably, Retrogenix Cell Microarray Off-Target Screening data were used to support the licensing application for Novartis’ Kymriah, the first CAR-T immunotherapy ever to receive FDA approval.
Case study: Covid-19 receptor identification
Applications of the Retrogenix Cell Microarray Technology are not limited to the analysis of off-target effects. In a recent case study performed together with Medicines Discovery Catapult and Peak Proteins in the UK, almost 6,000 human proteins were screened against the SARS-CoV-2 spike protein, which the Covid-19-causing virus needs to enter human cells.
Besides several previously known targets, three novel receptors were identified. Overall, the data suggest that SARS-CoV-2 may be a highly promiscuous virus exploiting multiple mechanisms of cell entry, contributing to its severity in specific patient groups.
“With this knowledge, we may further understand disease pathogenesis of Covid-19 patients and how infection with SARS-CoV-2 may lead to failure of specific organs. Understanding the binding profile of pathogens such as SARS-CoV-2 in greater detail will help in the discovery of new therapeutics, as well as the repurposing of existing drugs,” Brown stated.
Joining forces for successful end-to-end drug development
The Retrogenix Cell Microarray Technology supports drug development projects from early target identification and screening of potential lead candidates to data generation for IND-enabling studies. With the acquisition of Retrogenix, Charles River aims to further strengthen its integrated, end-to-end solution for therapeutic antibody and cell and gene therapy discovery and development.
“The long-standing experience of Charles River’s expert teams, such as their world-class IHC-based screening capabilities, is an excellent fit with the Retrogenix Cell Microarray Technology,” Brown said. “We are excited to join Charles River in supporting the development and approval of safe new therapies.”
Find out more about the Retrogenix Cell Microarray Technology, sign up to receive capability and library updates, and find out about forthcoming webinars.
All images courtesy of Charles River Laboratories