Safety, Efficacy Results Boost the Use of NKT Cells in Pediatric Neuroblastoma

Of the 11 patients treated, 2 achieved a partial response, 1 of whom is now in complete remission (CR) after receiving the second dose of CAR-NKT cell infusion, according to Andras A. Heczey, MD, lead study author and an assistant professor pediatric oncology at Baylor College of Medicine. In addition, 4 patients had stable disease and 5 experienced disease progression.

“CAR NKTs can be manufactured effectively on a clinical scale,” Heczey said. “These cells are safe and there is evidence of in vivo expansion and localization to metastatic sites. Now we are seeing tumor regression in 3 patients, including 1 durable CR.”

CAR T-cell therapy has shown good clinical results in multiple lymphoid malignancies, and this modality has changed the standard of care for many patients with these cancers. However, in the treatment of solid tumors, the strategies with these therapies still need to be refined.

“Maybe a way to improve [these strategies] is by looking at specific subsets of lymphocytes that may have better antitumor properties for solid tumors than general bulk expansion of -T cells,” Heczey said. “We believe NKT cells are such a subset.”

T cells recognize peptides presented by the major histocompatibility complex (MHC) class I and II molecules and have a variety of T cell receptors that allow them to do so. Unlike the T cells, NKT cells can recognize hydrophobic molecules, such as glycolipids, presented by the CD1d molecules. In addition, they express an invariant T cell receptor (iTCR) containing the V24 chain that is preferentially paired with the 11 chain, according to Heczey.

“We all share this iTCR in our NKT cells, which is important, because they can regulate the same processes in all of us,” Heczey explained. “As the field moves towards turnkey approaches, these NKTs have no potential for induced graft-vs-host disease.”

In terms of their potential efficacy, NKTs move in a chemokine-dependent manner to solid tumors, specifically to neuroblastoma, in a CCL2/CCL20-dependent manner. More importantly, according to Heczey, once at the tumor site, the NKTs target tumor-associated macrophages (TAMs) through a CD1d/iTCR interaction; CD1d is expressed on TAMs. Through this indirect mechanism, the infiltration of NKTs into neuroblastoma tissues has been associated with improved outcomes.

Preclinical data have shown that NKTs developed with a CAR expressing GD2 can target tumor cells directly and indirectly by destroying tumor-supporting TAMs; this has been demonstrated in neuroblastoma models.

These results prompted the launch of GINAKIT2 (NCT03294954), which aimed to investigate genetically engineered NKT cells expressing an optimized GD2-CAR and IL-15 to treat children with relapsed/refractory neuroblastoma in the autologous setting. . “IL-15 was included because this cytokine can enhance the persistence of expansion and, as a result, the antitumor properties of NKTs in the preclinical setting,” Heczey noted.

To be eligible to enroll, patients had to have confirmed high-risk relapse/refractory neuroblastoma, have a life expectancy of at least 12 weeks, and be between 1 and 21 years of age.

The study used a 3 + 3 dose-escalation design and patients were treated at 4 dose levels: dose level 1 of 3 × 106, dose level 2 of 1 × 107, dose level 3 of 3 × 107, and dose level 4 of 1 × 108. In addition, patients received a standard lymphodepletion regimen and this included cyclophosphamide given at a dose of 500 mg/m2 on days -4, –3 and –2, and intravenous fludarabine at a dose of 30 mg/m2 on days –4 and –3 to cell infusion.

On day 0, patients received the CAR-NKT cell infusion and were evaluated weekly in the outpatient setting. At week 2 post-infusion, patients had a biopsy; at week 4, they underwent imaging and evaluation of response to therapy.

“The safety assessment is focused on the first 28 days after the infusion,” Heczey added.

The primary endpoint of the study is safety, and other clinical endpoints include CAR-NKT cell persistence and trafficking, as well as antitumor responses.

Of the 11 patients enrolled and treated to date in the study, the median age was 7 years (range 2-12) and all had relapsed/refractory, high-risk stage IV neuroblastoma. In addition, all patients were able to receive the full prescribed lymphodepletion regimen and the full CAR-NKT cell infusion.

“NKTs are a small subset of peripheral lymphocytes … so the question was whether the right amount of NKTs could be generated ex vivo,” Heczey said. “So far we have been able to generate these products with high purity, up to 98% and sometimes higher, with nice CAR transduction efficiency as well.”

With this, the absolute number of NKTs, even in smaller children, is approaching 1 billion cells, according to Heczey. In the beginning, researchers planned these extensions for about 21 days before production. “Now we’re at 14 days or less and we’re still generating a large amount of very potent NKT cells,” Heczey said.

To measure product heterogeneity between patients and differences in antitumor properties, NKT cells were repeatedly exposed to tumor cells in a co-culture assay. The CAR-NKT cells were replated several times with neuroblasts during this process. Researchers then evaluated their ability to expand and maintain their cytolytic capacity and measured the expression of depletion markers.

“Most CAR-NKT products can maintain their ability to kill even after 5 rounds of co-culture,” Heczey noted. “However, there are some [in which] the ability to kill drops significantly by the fifth round. If you look at the folding extension, there are clear differences between these products. Perhaps the highest fold extension for our product is at the beginning, as we would expect.

Markers in the CAR-NKT products, differences were seen in proportion of TIM-3 and PD-13 dual-positive populations. This could indicate that those cells may be more depleted than others, Heczey explained.

After infusion, CAR-NKT cells expanded in the peripheral blood of patients. In addition, the absolute number of CAR-NKT cells varied significantly from patient to patient and did not appear to be dose level dependent. In all but 1 of the patients enrolled in the study, Heczey said the frequency of CAR-NKT cells was higher than before the infusion. In addition, CAR-NKT cells were detected in peripheral blood in all patients, with a peak expansion around week 2 and week 3 after infusion. In addition, at all dose levels, CAR-NKT cells were found in the neuroblastoma tumor sites, as well as in bone marrow biopsies.

In terms of GD2 expression in the CAR-NKT cells, 2 patients were found to have downregulated expression. Researchers believe this loss of expression may have occurred prior to enrolling to study, but there are no biopsies to confirm this, Heczey said.

In addition to CAR-NKT cell expansion, tumor burden, as measured by Curie scores, was also found to be associated with antitumor activity in patients; those with high area under the curve/Curie score values ​​were those who experienced antitumor activity.

In terms of safety, investigators have not observed dose-limiting toxicities in the first 28 days after CAR-NKT infusion. Most reported grade 3 or 4 adverse events (AEs) were mostly hematologic, which is consistent with what has been seen in patients who have received a cyclophosphamide/fludarabine lymphodepletion regimen, Heczey said. The most common Grade 4 adverse reactions were neutropenia (n = 10/11), lymphopenia (n = 6/11), leukopenia (n = 6/11), and thrombocytopenia (n = 2/11).


1. Heczey A, Courtney A, Lui K, et al. Natural killer T cells expressing a GD2-CAR and IL-15 are safe and can induce complete remission in children with relapsed neuroblastoma – a first-in- human, phase 1 study. Summary presented at: 2021 American Society of Gene & Cell Therapy Annual Meeting; May 11-14, 2021; Virtual. Summary 198.

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