Lab mice

Photo by Aaron Logan

Researchers say they have discovered a method for expanding natural killer T cells (NKTs) that ensures their persistence, thereby making NKTs more attractive as chimeric antigen receptor (CAR) carriers for cancer immunotherapy.

When transduced with a CD19-specific CAR, the researchers’ persistent NKTs produced sustained tumor regression in a mouse model of B-cell lymphoma.

The team described this work in the Journal of Clinical Investigation.

“NKT technology is quite powerful and offers a significant potential for treatment of cancer,” said study author Leonid Metelitsa, MD, PhD, of Baylor College of Medicine in Houston, Texas.

“But for it to be most effective, we have to find the best way to expand the cells ex vivo while preserving their ability to persist once delivered back to patients. If they can persist in the body for a long time, they have much longer therapeutic activity, and this is essential for fighting cancer.”

Molecule affects persistence

The researchers noted that central memory T cells are known for their ability to proliferate and persist, and these cells are characterized by expression of the surface molecule CD62L.

In this study, the team found that NKT cells freshly derived from blood did not express CD62L, or it was expressed at a very low level. However, after the researchers expanded NKT cells, they found that CD62L was expressed at higher levels.

“We consistently identified a subset of cells present at very high numbers after the first 12 days of expansion, and critical to this subset of cells was the presence of CD62L,” Dr Metelitsa said. “In fact, they became the majority of the new cells.”

In addition, Dr Metelitsa said that CD62L-positive NKT cells were responsible for further propagation of NKTs in culture, which is important for achieving large numbers of cells. However, extensive culture led to the eventual decline of CD62L expression in NKTs.

To test the role of CD62L in NKT-cell persistence, the researchers delivered CD62L-positive and CD62L-negative NKTs to immune-deficient NSG mice. They found that CD62L-positive NKTs persisted 5 times longer than CD62L-negative NKTs.

CAR-NKTs fight lymphoma

Next, the researchers transduced CD62L-positive and CD62L-negative NKTs with a CD19-specific CAR and delivered these cells to mice with B-cell lymphoma.

The team found that both CD62L-positve and CD62L-negative CAR-NKTs prolonged the survival of mice, when compared to controls (P<0.001).

However, only the CD62L-positive CAR-NKTs induced sustained tumor regression. Seven of 9 mice that received CD62L-positive CAR-NKTs lived, and 5 were tumor-free for at least 3 months. But all 10 mice that received CD62L-negative CAR-NKTs ultimately succumbed to tumor progression (P<0.001).

Costimulation improves NKTs/CAR-NKTs

The researchers then turned their focus to costimulation of NKTs in order to maintain the subset with a high percentage of CD62L-positive cells in prolonged culture. Costimulation involves the interaction of receptors on NKTs with activating molecules on an antigen-presenting cell to increase the NKTs’ immune functions.

“We have known that costimulation is an important part of immune response and immunotherapy, but, in this case, we did not know which costimulatory molecules would be important for the expansion and persistence of CD62L-positive NKT cells,” said Gengwen Tian, MD, of the Baylor College of Medicine.

After testing more than 100 combinations, the researchers discovered that combining an antigen-presenting molecule—CD1d—with 3 costimulatory molecules—CD86, 4-1BBL, and OX40L—induced prolonged persistence and better therapeutic activity of NKTs and CAR-NKTs in mouse models.

“When we developed an antigen-presenting cell clone that expressed CD1d with all of these costimulatory molecules at certain levels, NKT cells maintained a high percentage of CD62L even in a prolonged culture,” Dr Metelitsa said.

The researchers conducted in vivo testing of CAR-NKT cells that were expanded with the original method or with the costimulation method. And they found that costimulated cells had significantly higher therapeutic activity in mouse models of neuroblastoma and lymphoma.

“Our goal now is to optimize our NKT cell expansion protocol so that we can obtain FDA approval to initiate clinical trials,” Dr Metelitsa said.

Method may produce better CAR-NKTs