APHERESIS-ELUTRIATION PROGRAM FOR ADOPTIVE IMMUNOTHERAPY WITH AUTOLOGOUS ACTIVATED MONOCYTES IN CANCER-PATIENTS

Human blood monocytes (Mo) and monocyte-derived macrophages (M empty set) are known to be potent antitumor cytotoxic effector cells through activation with recombinant human interferon gamma (rIFN-gamma), bacterial muramyldipeptide or the synthetic derivative muramyltripeptide phosphatidylethanolamine entrapped in liposomes (L-MTP-PE). Large-scale generation of ex vivo activated Mo from the blood of cancer patients proved feasible. We report our experience with a fixed rotor speed counterflow centrifugation elutration (CEE) procedure using the newly available Beckman high capacity JE-5.0 rotor system that reproducibly isolates up to 1.0-1.5 x 10(9) Mo with > 90% purity, in suspension and functionally intact derived from peripheral blood mononuclear cell-enriched suspensions obtained by leukapheresis (LP) from healthy volunteers and cancer patients. The semiclosed, easy to handle CCE system, was adapted to a sterile technique that permitted clinical trials in adoptive monocyte immunotherapy. Freshly isolated Mo did not lose morphological or functional integrity and had no spontaneous activation. Their abilities to become activated to the cytotoxic state after 18-h stimulation with 500 U/ml rIFN-gamma or 1-mu-g/ml L-MTP-PE and to differentiate into matured M emtpy set in vitro were not altered The system was therefore used to isolate large numbers of Mo for a phase I clinical trial of intraperitoneal immunotherapy with L-MTP-PE activated autologous Mo in nine patients with peritoneal carcinomatosis. Each patient received weekly Mo infusions (n = 5) with an intrapatient dose escalation schedule (from 10(7) to 10(9) Mo). Toxicities were mild including fever, chills and abdominal pain. There was no treatment-induced thromboembolic event or capillary leak syndrome. The degree of Mo activation in vitro was shown by the release of procoagulant activity and monokines (IL-1, TNF-alpha and IL-6) in the culture supernatants and the antitumor activity against U937 cells and human ovarian carcinoma cells in vitro and in tumor-bearing nude mice in vivo. The in vivo immunomodulatory activity of i.p. infused Mo was demonstrated by an elevation of WBC and granulocyte counts, fibrinogen, C-reactive protein and neopterin in peripheral blood and generation of IL-1, TFN-alpha and IL-6 in peritoneal fluid. Phase II clinical trials, now in progress, should further define the therapeutic potential of human adoptive cellular immunotherapy using Mo or M empty set.