A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

Abstract

Background: Natural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancer.

Methods: Patients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m(2) × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometry.

Results: Twenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30-65) years. Mean NK cell dose was 2.16 × 10(7)cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = <0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansion.

Conclusions: Adoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.

Figure 1

Treg increase 14 days after NK + IL-2 administration. PBMC were collected prior to chemotherapy and 14 days after haplo-identical NK cell infusion and IL-2 administration. When all the ovarian patients were considered, there was a significant increase in Treg, but two patterns were seen. Seven patients exhibited a high Treg profi le, defined by the presence of CD4⁺, CD25⁺ and CD127^– cells at day +14. Six patients exhibited a low Treg pattern, where the post-therapy sample did not change from pre-therapy. There was no difference in Treg elevation based on presence or absence of TBI (P = 0.44). Error bars represent standard error of the mean.

Figure 2

Phenotypic Treg express Foxp3. PBMC were collected prior to chemotherapy and 14 days after haplo-identical NK cell infusion from a patient in the high Treg group. At day +14, 52.6% of circulating cells were CD3⁺ CD4⁺.Of those, 90% were consistent with circulating Treg. The surface phenotype panel for Treg CD25⁺ CD127^– correlated closely with the percentage of true Treg (95%), as defined by the expression of Foxp3.

Figure 3

IL-15 levels increase after lymphodepleting therapy. Plasma was collected from patients at the indicated time-points before and after the Hi-Cy/Flu ± TBI preparative regimen. A significant increase in IL-15 concentration was detected following the chemotherapy prior to donor NK infusion and at day + 7. By day +14 concentrations began to decrease. Error bars represent standard error of the mean.