Within the CAR-T cells themselves, the site at which the lentiviral vector integrated into the DNA during the engineering of the CAR-T cells was most commonly at introns and intergenic regions (noncoding regions). The vector integration site is typically unique for each clone, and by fate mapping the integration sites, the study researchers found that after the initial infusion, the first patient had more than 8000 unique vector integration sites and the second patient (who received a lower dose) had more than 3700 unique vector integration sites, suggesting an initially diverse population of CAR-T cells. Over time, the number of unique vector integration sites declined for both patients and it appeared that only a small number of clones persisted and maintained remissions.

“These types of deep dives into persistence in each patient — while they showed differences — hopefully they will eventually lead to better knowledge about how to make a better CAR-T cell and one that will persist and provide a lasting antitumor activity,” said Dr Maloney.

These deep dive observations have also sparked questions. For instance, how can CD19 CAR-T cells persist in the absence of the tumor antigen? Normal B cells express the target antigen, and that “most likely drives the high level persistence of these CAR-T cells,” explained Dr Melenhorst. “The fact that we do not see [B] cells in the circulation bears witness to the efficient elimination of those cells by CAR-T cells.”

The vector integration site observations in particular brought forth the question of whether CAR-T cell potency is linked to a particular vector integration site. Dr Melenhorst and colleagues previously characterized another patient who also had a vector insertion into a particular gene and saw an enhanced potency of the CAR-T cells. 

“Building off of that observation, we now have in these patients the long-term persistence of their CAR-T cells and we also see the same integration sites recurring again and again,” Dr Melenhorst said. 

A more detailed analysis of the specific vector integration sites will be revealed in a forthcoming manuscript. 

Dr Melenhorst noted that, at this point, researchers cannot control the location of the CAR-T integration site — and similarly, might not be able to control CAR-T cell potency — with lentiviral vectors. “This is an area of active investigation in my lab and many others,” he said.

“What we really don’t know though is whether these cells are absolutely critical to keeping these patients in complete remission,” said Dr Maloney. “We don’t know [whether CAR-T cells are critical] because normal B cells also trigger these cells to persist.” He added, “We don’t really know that these cells are continuing to keep the CLL in remission or whether the underlying CLL has actually been cured by the treatment.”

References

  1. Melenhorst JJ, Porter DL, Nobles CL, et al. Long-term remission of CLL sustained by oligoclonal CD19-specific chimeric antigen receptor T cell clones. Presented at: 2019 American Society of Gene and Cell Therapy (ASGCT) Annual Meeting; Washington, D.C.; April 29-May 2, 2019. Abstract 358.
  2. Porter DL, Hwang WT, Frey NV, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7(303):303ra139.
  3. Porter DL, Levine BL, Kalos M, Bagg A, and June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365(8):725-733.