New Microscopy Technique Challenges Therapeutic Antibody Classification

In the 1990s, researchers developed the first monoclonal antibody (mAb), trastuzumab (Herceptin), which works by blocking a receptor that is overexpressed in cancer cells.¹ This development was revolutionary for the field of immunotherapy. Since then, mAbs have been widely used to target specific molecules on tumor cells and trigger cell death. However, researchers do not completely understand the molecular mechanisms by which mAbs bind to their target and how this activates the immune system to attack cells.

Now, researchers have developed a new super-resolution microscopy technique to visualize the molecular interactions between therapeutic mAbs and their target receptors on tumor cells.² Their findings, published in Science, demonstrate how advanced imaging techniques can provide deeper insights into the molecular mechanisms of immunotherapy, paving the way for improved treatments for patients.

“There is a power in seeing things,” said Pratik Kumar, a chemical biologist at the National Center for Biological Sciences, who was not involved in the study. “Seeing how the antibody binds and [causes changes] over time is very interesting.”

The seeds for this research were sown about 10 years ago, when Markus Sauer, a biophysicist at the University of Würzburg started a collaboration with clinicians at the University Hospital Würzburg. Together, they explored the utility of super-resolution microscopy, a technique that Sauer had been using in the field of neurobiology and immunology, in characterizing tumor antigens.

Applying this technique helped them quantify antigens at a much higher sensitivity than conventional flow cytometry methods, highlighting the power of super-resolution microscopy in the field of immunotherapy.³

This prompted them to investigate further uses of super-resolution microscopy in immunotherapy. “We thought maybe this is a method to shed some light on the mode of action of these [monoclonal] antibodies,” said Sauer.

Sauer and his team started out by improving on a technique called DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT).⁴ The technique, which images samples at a 10 nanometer-resolution in two dimensions (2D), uses a single-stranded DNA attached to an antibody that binds to a cellular target. A fluorescently-labeled complementary strand binds and unbinds to the DNA, which results in blinking signals.

However, even when unbound the complementary DNA strand emits a fluorescent signal, giving rise to background noise. As a result, researchers must use limited concentrations of the labeled DNA, which results in a weak signal and imaging times as high as 20 hours per cell.

To improve this, Sauer and his team added two fluorescent dyes to the complementary DNA. This caused the formation of a dimer in the unbound state, reducing fluorescence. “So now you have virtually zero background,” explained Arindam Ghosh a biophysicist at the University of New South Wales and study coauthor. “In this way, we can use higher concentrations of these [DNA strands] now to speed up the process.”

As evidence of the improved DNA-PAINT technique, the researchers were able to image microtubules at a high resolution in just five minutes, as opposed to an hour with the original method. To enable 3D imaging, which would be essential for understanding how mAbs bind to receptors, they combined the modified method with a technique called lattice light-sheet (LLS) microscopy, which uses ultrathin light sheets to scan planes, which can be assembled to create a 3D image. “[Then], we could get the whole cell volume within four- to four-and-a-half-hours,” said Ghosh.

Equipped with a 3D super-resolution microscopy technique, Sauer and his team set out to investigate how mAbs bind to the B cell surface receptor CD20, a common immunotherapy target for some blood cancers. They started by treating human lymphoma cells with one of four different mAbs, classified as type I or II based on how they interact with CD20 and induce cell-death: Type I mAbs crosslink CD20 triggering the immune system’s complement pathway, while type II mAbs do not.⁵However, when they looked under the microscope, they saw something that would challenge this classification system.

Sauer and his team found that, irrespective of their class, every mAb they tested crosslinked CD20 molecules, leading to mAb accumulation on the B cell surface. This provided a site for complement system proteins to bind, which triggers a cascade of immune reactions.

“Type II mAbs had been described as not crosslinking CD20 and using other killing mechanisms [than complement activation],” said Sauer. “It was a really big surprise to us that all the antibodies tested crosslinked CD20.”

The researchers also observed that CD20 molecules were present on finger-like projections called microvilli on B cell surfaces. Images taken over time revealed that mAb binding led to an asymmetric distribution of the microvilli towards one side of the cell, resembling a hedgehog, where there is a smooth side and a spiky side. Live cell imaging using labeled mAbs and LLS microscopy further validated these results, highlighting that the approach can be used to study how antibodies bind to their receptors at a high resolution.

Such a method can help researchers better understand how therapeutic antibodies work, said Kumar, and is an improvement over the current methods because of a shorter imaging time. “But it still takes time if you want to go through multiple cells,” he said, adding that future work can focus on further reducing the noise to allow for even faster imaging. Nevertheless, he noted, “This is a step in the right direction.”

Investigating how cells obtained from patients differentially respond to mAbs can help the researchers understand why some patients do not respond to immunotherapy, said Sauer. He added that for now, the team is focusing on understanding what happens once the mAbs crosslink CD20. “What we are now working on is to visualize [how] the follow up process is like.”

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