A Phase I Trial of Adoptively Transferred Ex-Vivo Expanded Autologous Natural Killer (NK) Cells Following Treatment with Bortezomib to Sensitize Tumors to NK Cell Cytotoxicity
Authors: Reger Robert N , Berg Maria , Lundqvist Andreas , Donohue Theresa , Carlsten Mattias , Betters Dawn M , Cook Lisa , Ramos Catalina , Grasmeder Sophia , Su Su , Stroncek David , Pantin Jeremy , Keyvanfar Keyvan , Carvallo Cristian A. , Khuu Hahn , Childs Richard 
Published: October 2019

Abstract
Abstract 1001
The proteosome inhibitor bortezomib sensitizes tumors in-vitro and in-vivo to autologous NK cell killing by augmenting NK cell TRAIL and perforin/granzyme-mediated caspase-8 activity (Lundqvist et al, Blood 2009). This effect occurs independent of tumor MHC class I expression, suggesting drug-induced tumor sensitization to autologous NK cell killing could be used to override the dominant inhibitory signaling that occurs via KIRs. Based on preclinical data, we initiated a phase I clinical trial to explore the safety and antitumor efficacy of escalating doses of adoptively infused ex-vivo expanded autologous NK cells following bortezomib treatment in patients (pts) with a variety of advanced malignancies refractory to conventional therapy. Pts underwent a 15–20L apheresis to isolate NK cells that were enriched using Miltenyi immuno-magnetic beads to deplete CD3+ T cells followed by CD56+ selection. Enriched NK cells (5–12 × 107cells) were expanded ex-vivo over 14–27 days using an irradiated clinical grade EBV-LCL feeder cell line. On day −3, pts receive a single injection of pentostatin (4mg/m2) to deplete Tregs followed by an injection of bortezomib (1.3 mg/m2) on day −1 to sensitize tumors to NK cell killing. Cohorts 1–4 received a single infusion of ex-vivo expanded NK cells on day 0 in a dose escalating fashion (5×106, 1×107, 5×107, and 1×108 NK cells/kg; 3–6 pts per cohort). Cohorts 5–6 received 1 × 108 NK cells/kg on day 0 and a second escalating dose of NK cells infused on day +5 (5 × 107 and 1 × 108 NK cells/kg respectively) following treatment with a second dose of bortezomib given on day +4. To maintain NK cell viability and TRAIL surface expression, 2 million IU/m2 of IL-2 was given s.c. every 12 hrs on days 0 through +6 in cohorts 1–4 and days 0 through +9 for cohorts 5–6. Pts with stable disease or regression were eligible to receive additional cycles of therapy. Twenty pts received a total of 73 adoptive NK cell infusions. 58/59 (98%) NK cell cultures expanded successfully to achieve the target NK cell dose. NK cells harvested 14–27 days after expansion contained a median 99.7% (range 92–100) CD3-/CD56+ NK cells and had a median 87% (range 71–93) viability.
NK cells for the first infusion given on day 0 expanded a median 199 fold (range 58–6647) ex-vivo after a median 14 days of culture (range 14–22). NK cells given on day +5 expanded a median 1298 fold (range 243-20, 196) after a median 20 days of culture (range 19–27). For cohorts 3–4, NK cells peaked in circulation at a median 382cells/μL (range 60–1851) at median 7 days following adoptive transfer. For cohorts 5–6, NK cells increased in the circulation a median 6.0 fold (range 1.4–7.0) over baseline, peaking at a median 266 cells/μL (range 61–301) at a median 10 days following adoptive transfer.
No grade II–IV toxicities related to NK cell transfer were observed. The most common adverse events were attributed to IL-2 therapy including grades I-II fever, renal insufficiency, edema and hypotension. Four pts developed elevated free T4 levels and low TSH levels following NK cell therapy consistent with acute thyroiditis; two became hypothyroid and required thyroid replacement therapy. Best clinical response to date in the first 20 pts treated included 6 pts with progressive disease, 10 pts with stable disease (including 2 pts with metastatic tumors who had more than a 30% decline in serum tumor markers) and 4 pts with a minor response (2 pts with renal cell carcinoma (RCC) and 2 pts with chronic lymphocytic leukemia (CLL)). Thirteen of 20 pts (66%) went on to receive more than 1 NK cell infusion including 1 pt who received 6 cycles, 3 pts who received 5 cycles, 4 pts who received 4 cycles, 3 pts who received 3 cycles and 1 pt who received 2 cycles before going off study for either progressive disease or personal preference.
In conclusion, this study has established that 2 infusions of ex-vivo expanded autologous NK cells at a dose of 1 × 108 cells/kg given on days 0 and +5 are safe with preliminary evidence for antitumor immunity being observed against metastatic RCC and treatment refractory CLL. With the exception of thyroiditis, infusions of ex-vivo expanded NK cells were well tolerated with no grade III/IV toxicities observed to date. This phase I study continues to accrue pts with cohorts 7–10 intended to establish the maximum tolerated dose of ex-vivo expanded NK cells that can be infused on day 5 (up to a dose of 1 × 109 NK cells/kg).

Disclosures:
No relevant conflicts of interest to declare.