A preprint reports that a single substitution in CD27 enhanced anti-CD70 CAR-T cells in cell culture and mouse experiments

A preprint reports that a single substitution in CD27 enhanced anti-CD70 CAR-T cells in cell culture and mouse experiments

In a preprint published on September 10, the authors mutated a single amino acid in CD27, the natural binding partner of the tumor-associated protein CD70. Computational screening narrowed the candidate space, and N88A then stood out in cell culture and mouse experiments.

CAR-T cells are made from T lymphocytes, cells of the immune system, by equipping them with a receptor that recognizes a chosen target. The recognition domain of such a receptor is usually derived from an antibody. In this work, the recognition domain was CD27 itself, the natural partner of CD70, a protein found on the surface of certain tumors.

In a 2025 study, some of the present authors had already compared such CAR-T cells in a mouse model of multiple myeloma, a tumor of plasma cells. A construct in which the recognition domain consisted of the entire extracellular fragment of CD27 expanded roughly 80 to 100 times more vigorously in mouse blood than the antibody-based variants tested alongside it. The current work asks whether CD27 itself can be improved.

The researchers took the structure of the CD27:CD70 pair and screened 24 amino acids at the binding interface using protein design software. Each receptor variant was inserted into the same genomic locus so that cells would produce it at roughly equal levels. N88A, a substitution of asparagine with alanine at position 88 of CD27, was computationally predicted to weaken binding; the experimental result was the opposite.

In co-cultures with two acute myeloid leukemia cell lines, N88A killed tumor cells faster. In a mouse leukemia model, all animals first received tumor cells and then, four days later, half a million CAR-T cells. In mice that received N88A, the tumor remained below the detection threshold through the end of the 85-day observation period; in the group that received the original CD27-CAR, median survival was 38 days. In a myeloma model, both CD27 versions held the tumor below the detection threshold by the end of the experiment, but N88A expanded more vigorously in the blood.

Experiments with purified proteins showed that N88A associated with CD70 roughly twice as slowly but dissociated more than four times as slowly. The resulting CD27:CD70 complex was therefore roughly twice as stable. A computational model suggests that the CD27:CD70 pair can adopt several bound conformations; this is consistent with the measured slow dissociation.

The authors also screened for off-target binding of the modified CD27. Besides CD70, a signal appeared for FCGR3A, a protein that binds the Fc laboratory tag; the same signal was produced by a control protein carrying that tag. In an assay with blood progenitor cells, the number of resulting cell colonies did not differ from the control.