Photo from Penn Medicine
New research has provided an explanation for resistance to CTL019, a CD19 chimeric antigen receptor (CAR) T-cell therapy.
Investigators analyzed samples from children with B-cell acute lymphoblastic leukemia (B-ALL) and found evidence to suggest that CTL019 resistance can be caused by CD19 splicing alterations.
These alterations prompt the loss of certain parts of the CD19 protein that are recognized by the CAR T cells.
The team described this work in Cancer Discovery.
They noted that 10% to 20% of B-ALL patients treated with CD19-directed immunotherapy may experience relapse.
“Some of them can be successfully retreated, but, in others, a more pernicious kind of leukemia may emerge, which no longer responds to CTL019,” said study author Andrei Thomas-Tikhonenko, PhD, of the University of Pennsylvania in Philadelphia.
“In some cases, resistance is accompanied by the disappearance of the target CD19 protein from the cell surface . . . . Our goal was to figure out how the CD19 protein manages to vanish and whether it is gone for good or whether it could, under certain circumstances, be coaxed back.”
“Our initial finding from this study was that, in most cases, the CD19 genetic code was not irretrievably lost. We also discovered that the CD19 protein was still being made, but as a shorter version, which escapes detection by the immune system.”
To understand the mechanism of CTL019 resistance, Dr Thomas-Tikhonenko and his colleagues studied multiple tumor samples from 4 children with B-ALL. The samples were collected before the patients were treated with CTL019 and/or after they developed resistance to the therapy.
The investigators found that, in some cases, 1 copy of the gene coding for CD19 (located on chromosome 16) was deleted, and the other copy was damaged as a result of mutations in coding areas of the CD19 gene, most frequently in exon 2.
However, the team also discovered alternatively spliced CD19 messenger RNA species in which exons 2, 5, and 6 were frequently skipped, making mutations in exon 2 largely irrelevant.
Subsequent investigation revealed that deletion of exons 5 and 6 resulted in premature termination of CD19.
Deletion of exon 2 resulted in the production of a modified version of CD19, which was more stable than its standard version. The shortened protein was functional and could perform many of the tasks that CD19 is known to handle, but it cannot be targeted by CTL019.
The importance of exon skipping in CTL019 resistance cannot be overstated, Dr Thomas-Tikhonenko said.
“Without exons 5 and 6, the CD19 protein has no way of being retained on the cell surface,” he explained. “The case of missing exon 2 is more complex. Although the resultant protein can make it to the cell surface, albeit not very efficiently, it can no longer be recognized by CTL019.”
He and his colleagues believe this research can inform future use of CTL019 and immunotherapy in general.
“[A]lternative splicing could be a potent, built-in mechanism of resistance, and it might be better to target proteins that, unlike CD19, are not prone to exon skipping,” Dr Thomas-Tikhonenko said.
“[In addition,] it might be important to preselect patients for CTL019 and similar therapies and make sure that the alternatively spliced CD19 variants are not already present in their leukemias. If they are, resistance could develop very quickly.”
Designing new immunotherapeutics that can recognize the shortened version of CD19 is another approach to overcoming CTL019 resistance, he added.
He and his colleagues noted that this study was limited by the relatively small number of samples analyzed, which might have prevented the investigators from identifying additional mechanisms of resistance.
