A Status Report on Cell-Based Protein Arrays

A review article from Charles River’s Retrogenix^® business and its pharma partners pushes the envelope of what’s possible with cell-based protein array technology

Whether you swallow a statin or receive a chemo injection, all drugs need to reach the right target. How this happens depends, in part, on three-dimensional proteins that lie on the surface of cells. These cells, called receptors, are what drug of any size needs to latch on to in order to get inside a cell and do its business.

Let’s say, for instance, you are a therapeutic antibody designed to target certain kinds of lung tumors. To do so, you need to find the right receptor on those tumor cells that allows the antibody to fit precisely to attach to it, like a key in a lock. 

Drug developers use different methods to confirm if their candidate is binding the correct target. The more traditional method is a tissue cross-reactivity (TCR) assay, which determines whether a drug article can bind to various human or animal tissues. But there is also an in vitro method known as cell-based protein array that provides an alternative and/or complement to TCR. Cell-based protein arrays also help to reduce the use of laboratory animals by weeding out, early on, poor-performing drug candidates.

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Despite its obvious advantages, not all companies are familiar with using cell-based protein arrays, which motivated scientists developing the Retrogenix^® platform, a Charles River acquisition and a pioneer in cell microarray technology, to pair up with four of the world’s largest pharma companies to generate an overview of the platform in the context of TCR and off-target assessment studies. The review appeared in last month’s issue of Toxicologic Pathology.

Eureka recently caught up with Nick Brown, one of the authors of the review. Nick leads the Client Services Team associated with the Retrogenix^® platform and is responsible for growing the technology’s capabilities and business portfolio globally.  This Q&A is part of Eureka’s occasional series Research Notes, which focuses on recently published scientific findings of our scientists.

For our readers who are not scientists, what exactly are cell-based protein arrays?

Nick: Cell-based protein arrays are an in vitro method of representing a large number of proteins in the context of a cell rather than just individually on its own. This can then inform on what a therapeutic molecule is interacting with, and by extension how it works and whether any safety risks may be present due to unexpected binding. A good way to think about it is to begin with more traditional protein arrays, which are essentially recombinant proteins captured on a chip. A cell-based protein array overexpresses these proteins on the surface of a cell, which means they're superior in terms of the proteins’ physiological relevance, and also their folding and representation. So, when you test a drug/therapy over the top of them and look for binding, the responses should be significantly more informative and relevant than taking a traditional protein array-only approach. 

How long have cell-based protein arrays been available commercially?

Nick: The Retrogenix^® platform is the oldest that we know, so it is the first-in-class and remains the gold standard in our opinion due to library coverage and data output quality. This platform has been in development for over 16 years, and it has continuously advanced in terms of the size and quality of the protein library, the versatility of drug type that can be screened, and the reproducibility of the results.

How many proteins sit on the surface of a cell?

Nick: So, there’s probably between 6,000 and 7,000 for human cells depending on how you classify. There are over 6,500 proteins represented in the Retrogenix^® platform library, though that includes proteins that are on the surface, and secreted proteins that are floating in the extracellular space as well. These secreted proteins are important for determining off-target risk levels, so we include these in our array by anchoring them to the cell surface. We essentially attach a little tether to them and to the cell, and the protein is captured ready to be tested against. 

What exactly do we mean when we talk about off-target effects?

Nick: Let’s say you have a therapeutic antibody; that would typically be designed and raised against a single target, or if it's a multi-specific molecule, it'll have multiple targets. This means it should only hit that protein target and cause the drug effects of interest, such as blocking something or activating something, or highlighting a pathogen. When a molecule has an off-target effect, it means it's hitting a target, a protein, or an element in the body that it shouldn't be, as well as the primary targets. And, of course, that can then lead to unexpected toxicity or side effects. So, for example, if it's hitting an off target that is highly expressed in heart tissue, it could obviously have huge toxicological considerations for that therapeutic. And as you have a more aggressive therapeutic, the risk level to normal tissues goes up. 

Is the key reason we use cell-based protein assays to help identify off-target effects? 

Nick: At this point, yes. The majority of the work we do with this platform is off-target analysis, so essentially taking a molecule that we know to have a primary target and looking to see if it hits anything else, which of course can inform on the risk. In this way it's a powerful de-risking tool. It's a way of screening against a very large library of proteins in a highly relevant way without needing human tissue in vivo studies. 

Are cell-based protein arrays becoming the preferred choice over tissue cross-reactivity assays? 

Nick: It's an interesting question with a nuanced answer. What I will say is tissue cross-reactivity has been the gold standard for this sort of analysis for over 20, 30 years. Cell-based protein arrays, and the Retrogenix^® platform in particular, are increasingly being used both as a complement to tissue cross-reactivity, and in some cases as an alternative to tissue cross-reactivity. For instance, there are some therapeutic molecules that are not compatible with immunohistochemistry-based techniques like tissue cross-reactivity. You can’t run a TCR study for a whole cell-based therapy, whereas you can assess specificity with the Retrogenix^® platform. So, what we are advising is that cell-based protein arrays are very good at identifying what protein a drug is binding, while the TCR assay is better at answering the “where” question – so, which tissues, which locations.

You also have more histological context with the TCR study. Some Sponsors may prefer to run one or the other depending on the situation, though in combination these technologies provide a very powerful picture for Sponsors. Charles River is the only company in the world able to offer both technologies under a single roof.

For the cell-based protein arrays, are they all human proteins expressed in human cells?

Nick: Yes, though we have also developed non-human arrays, too, which ultimately will help us to figure out a therapy’s binding characteristics across a range of species and guide relevant tox-species selection decisions.

Is the primary 3Rs benefit reduction?

Nick: While I think replacement is the ultimate future goal, right now this technology is helping to reduce and refine the use of animals by functioning as a de-risking screen to help identify and filter out non-viable candidates, and to inform of toxicology species section decisions. You can answer a lot of questions that previously might have required an animal model.

Let's talk about the review article.  Who were our partners on this project and what was the objective? 

Nick:  So aside from Charles River, the co-authors included colleagues from Boehringer-Ingelheim, Novartis, Janssen, and Takeda. We have been working with these clients for many years, and they have a lot of faith in these platforms. I think they are keen on trying to advance the conversation to improve the speed, efficacy and safety of drug development, and push the envelope of what’s possible with this type of technology. 

A key takeaway from this article is that TCR has been the gold standard for many years, and referenced in a lot of the regulatory guidelines, though there have been recent updates to those guidelines that soften that approach and allow for either TCR or additional or suitable alternative methodologies. So, we are trying to build up a bit more confidence in cell-based protein arrays as a compliment or alternative, able to provide specific and actionable off-target information to get therapeutics to patients more quickly and safely.

There are no real silver bullets in science. Were there any caveats you found in reviewing the research that are worth mentioning with regard to cell-based protein arrays?

Nick: There's pros and cons to each technique. As I said earlier, one of the pros with cell-based protein array is that you get specific data, whereas for TCR, there can be difficulties interpreting what you see sometimes. I think the main advantage of TCR is that you're essentially seeing the therapeutic interacting with a true piece of tissue, which is more physiologically-relevant than a cell-based protein array using a recombinant overexpressed system. So, that's why, again, there are situations where it would be useful to use both, for example depending on the potential toxicity risk profile of the therapeutic agent, and Charles River can help Sponsors here.