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Michael Marand
From art to sports and even scientific research, a small adjustment in just the right place can have large reverberating benefits. In this theme, a team from the University of Pennsylvania recently shared their work in which modifying a single amino acid in a large protein revealed a potential immune therapy that could be useful against nearly all types of blood cancers.
CAR-T therapies have emerged as a promising approach to treat several blood cancers. Naturally occurring T cells protect against infection and cancer by mounting attacks against diseased cells that express specific signals on the cell surface. However, cancers can hide these signals to evade T cells. CAR-T therapy involves collecting T cells from a patient and engineering them in the laboratory so they express chimeric antigen receptors (CARs) that allow the T cells to better recognize cancer cells. Once the CAR-T cells bind to their target proteins, they become activated. They multiply and release signaling molecules that initiate the killing of the cancer cell. Since 2017, there have been six FDA approvals in CAR-T therapies for blood cancers, each of which is specific for one or two types of lymphoma, leukemia, or multiple myeloma. Yet, there remain other blood cancers that have proven much more difficult to treat with immunotherapy. The limiting factor in many cases is the identification of a target protein that is present on the cancer cells but absent, or rarely present, in healthy blood cells. Such a target allows the CAR-T therapy to kill cancer cells without killing healthy cells and causing adverse side effects.
A recent study aimed to develop a CAR-T therapy that can target any type of blood cancer, rather than focusing on one type at a time. To do this, the team used CAR-T cells that are targeted to the CD45 protein. CD45 is one of the most commonly expressed cell surface proteins across human blood cells. In this study, the researchers detected CD45 on 21 out of 22 different blood cancer cell lines, spanning leukemia, lymphoma, and myeloma categories. This means that a CAR-T therapy that targets CD45 could theoretically be useful in treating all types of blood cancers.
However, because CD45 is so universally expressed, the CAR-T cells themselves express CD45 as well. This means CD45-targeted CAR-T cells would likely kill fellow CAR-T cells in a process known as fratricide. While we do have technology that could remove the gene that encodes for CD45 protein in the CAR-T cells, thereby protecting them from themselves, this is not a feasible path. CD45 is essential for cell functionality, so eliminating CD45 altogether from the CAR-T cells diminishes their activity. Instead, the team sought to slightly modify the CD45 protein on CAR-T cells so that the CD45 could still carry out its essential function, yet it would not be recognized as a binding site by other CAR-T cells.
To block binding to CD45, it was first necessary to find out where on the CD45 protein CAR-T cell receptors bind. To find the general binding site, a series of mutant CAR-T cells were made, each one with an increasingly large portion of the protein cut off. CAR-T cells were still able to bind to mutant CAR-Ts with truncated CD45 up until a particular region called the D1 domain was deleted. This, of course, suggests that the CAR-T cells recognize the D1 domain in order to bind. Next, the team used CRISPR-based editing technology to modify specific amino acids with a non-reactive amino acid called alanine and then observed the effects on CAR-T cell activation. This revealed a few amino acids that were crucial to binding, all clustered in the same portion of the D1 domain.
Next, mutant CAR-T cells were made with a single amino acid modification. This small change was sufficient to protect the CAR-T cells from being killed by CD45 CAR-T cells. Importantly, unlike the CAR-T cells in which CD45 was knocked out entirely, the CAR-T cells with modified CD45 retained their function.
You may have already recognized the major problem remaining with this CD45 CAR-T therapy. CD45 is so common that tumor cells are not the only ones that express the target protein for this CAR-T therapy. Hematopoietic stem cells (HSCs), which give rise to various healthy blood cells, also express CD45, making them a potential unintended target of the CAR-T therapy. The team aimed to modify the CD45 on HSCs so that they could hide from the CAR-T cells, just as they had done to prevent the CAR-T cells from targeting each other. When the team introduced these modified HSCs and then dosed them with CD45 CAR-T cells, as expected, the unedited HSCs were eliminated, but in their place, the modified HSCs multiplied and repopulated. Just like a swiftly changing trend in clothing fashion, modified CD45 was suddenly ‘in’ with the HSCs, and the boring old unmodified CD45 was out!
To test the tumor-treating efficacy of the CD45-modified CAR-T cells, the CAR-T cells were introduced into the bloodstream of mice with a particularly difficult to treat cancer called acute myeloid leukemia (AML). The concentration of AML cells in the blood of mice treated with CAR-T cells decreased to nearly undetectable levels over the course of the experiment, while the AML concentration increased in 3 out of 4 control group mice. Months later, the researchers reintroduced more AML tumor cells into the mice that had survived the initial tumor challenge. These mice remained tumor-free, indicating that the original CAR-T cells had remained alive and capable of activation.
Finally, putting it all together, the team tested the CD45-modified CAR-T cells in combination with modified HSCs in mouse models of AML. In the small study, the 9 mice receiving the therapy had a significant survival benefit over the 4 control group mice. And the modified HSCs replaced the natural, unedited HSCs, as expected.
Overall, the authors have put forth an intriguing case for a universal blood cancer therapy that could help treat many kinds of blood cancers while sparing healthy cells. Key to the entire approach is the concept of editing the binding site that the CAR-T cells recognize. Even modifying a single amino acid appeared to be sufficient. This is particularly remarkable when we consider that the CD45 protein is made up of about 700 amino acids in all. The selection of CD45 was a wise one, even beyond its ubiquitousness across different types of blood cancers. Research suggests that CD45 is essential for leukemia growth and maintenance. This means that cancer cells are unlikely to decrease the expression of CD45 in order to evade this CAR-T therapy.
The authors emphasize that further investigation is needed to understand the safety of editing the CD45 binding site on HSCs. Additionally, the amino acid sequence of the binding site is different between mice and humans, so a follow-up study in nonhuman primates will be telling.
Edited by Mia Hubert
Works Discussed:
Car T cells: Engineering immune cells to treat cancer. National Cancer Institute. Accessed November 12, 2023. https://www.cancer.gov/about-cancer/treatment/research/car-t-cells.
Wellhausen N, O’Connell RP, Lesch S, et al. Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy. Science Translational Medicine. 2023;15(714). doi:10.1126/scitranslmed.adi1145
Figure created with Biorender
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