Donor identification was performed among patients’ relative in a first step. In the absence of familial donor, selection was performed among healthy blood donors from the blood bank. At least one KIRL (Killer Immunoglobulin Receptor Ligand) mismatch was required. MHC class I alleles considered for donor selection were HLA A3-A11, HLA-Cw or HLA Bw4. A two digits HLA genotyping assay was used to determine donor and patient HLA A3-A11, Cw or Bw4 status. High- resolution genotyping was performed to confirm HLA allelic expression.
Allogeneic NK cells production
Peripheral blood mononuclear cells (PBMC) from selected healthy volunteer’s donors were isolated by leukapheresis. CD3C T cells were depleted using anti-CD3 magnetic microbeads. Meanwhile, plasma was produced from a 100 mL blood sample taken from the donor. T cell depleted mononuclear cells were then washed and cultured overnight with 10% autologous sera and 1000 IU/mL of IL-2 in X-VIVO 15 medium.
Before adoptive transfer, cells were washed two times and suspended at 5.106/mL in albumin 4%.
Patients were treated with lymphodepleting chemotherapy (fludarabine 25 mg/m2 and cyclophosphamide 60 mg/kg) before NK cells injection. After overnight interleukin-2 activation, the cell therapy product was immediately infused through the hepatic artery under radiological guidance by the interventional radiologist. Allogeneic NK cells were adoptively transferred according to a dose escalation protocol,classical 3C3 design. The three escalating dose levels were 3.106, 8.106 and 12.106 NK cells/kg of recipient body weight. IL-2 (10.106 IU) was administered subcutaneously, starting 6 hours after the adoptive transfer and every 3 days for 6 injections. The anti-EGFR monoclonal anti- body Cetuximab (250 mg/m2) wasadministered intravenously every week for 7 weeks, starting from day 1. The patients were kept under observation for 24 h post procedure and discharged on the subsequent day. During the hospital stay, all clinical parameters and adverse events were recorded.
Neutropenia was observed in all patients, with a mean duration of 6 days (1-9 days). Two patients had febrile aplasia requiring a short-term antibiotherapy. Grade 3/4 anemia and thrombopenia were also described but had only little impact since red blood cell transfusion was only prescribed in one patient. Acute graft versus host disease was recently reported in patients receiving IL-15/4-1BBL activated NK cells following T-cell depleted stem cell transplantation but did not observe any adverse event related to the low residual allogeneic T lymphocytes. As NK cell infusion was intra-arterially infused, liver toxicities were also monitored. No grade 3/4 toxicities were reported based on ASAT, ALAT or Alkaline phosphatase concentrations. Two patients presented an increase of GGT after cell infusion however their GGT values were subnormal prior enrolment. Furthermore, any severe adverse effects associated to high dose of interleukin-2 (IL-2) and Cetuximab treatments following the NK cell transfer was reported in these patients. Altogether, in situ allogeneic NK cells infusion in liver metastasis plus systemic Cetuximab was well tolerated and no dose limiting toxicity was reported.
One partial response was observed in a colorectal cancer patient who was injected with 8.106/kg NK. This patient had only a liver metastatic disease, a KRAS exon 2 wild type colorectal cancer and never responded to previous therapies including anti- EGFR. FDG-PET scan assessment confirmed a decrease of liver metastases metabolic activity in most lesions after NK cells transfer.
Another patient with 3 liver metastases of a pancreatic adenocarcinoma experienced a dissociated response with the progression of two lesions and the complete regression of the smallest lesion in the right liver. Two additional patients with colorectal cancer experienced stable disease. However, all patients had progressive diseases 4 months following study enrollment. A stable disease was observed in patients receiving a cell therapy product with one KIR ligand mismatch. One partial response, a dissociated response and a stable disease occurred in the 5 patients treated with NK cell products harboring 2 KIR ligand mis- matches.
This result demonstrated that combining allogeneic NK cells transfer via intra-hepatic artery, cetuximab and a high- dose IL-2 is feasible, well tolerated and may result in clinical responses for patients with gastrointestinal carcinoma with liver metastasis.
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Scientific article publishing date : 1/3/2018
Immucura identifier BSC21_192EN