Long-lasting in vivo cytolytic response of NK cells against HSP70-positive tumors

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Introduction: Incubation of NK cells with the HSP70-peptide TKD (multimmune GmbH, Munich) plus low-dose IL-2 (TKD/IL-2) stimulate the cytolytic activity of resting NK cells against HSP70 expressing tumors in vitro and in tumor mouse models. In a phase I clinical trial we demonstrated that up to 6 reinfusions of TKD/IL-2-activated autologous NK cells are well tolerated, feasible and initiate immunological responses, i.e. enhanced cytolytic activity of NK cells and enhanced CD94 cell surface density. Patient: we investigated the magnitude and kinetics of mobilisation and the specific antitumor activity of NK cells in one patient receiving 6 + 3 reinfusion cycles of ex vivo TKD/IL-2-activated autologous NK cells. This patient came to our attention as of time of an anastomosis-relapse of a colon carcinoma diagnosed and resected 2 years before (as pT3, N2, M0, G2). The patient had refused adjuvant chemotherapy. After surgery, a biopsy of the colon carcinoma relapse was provided to our laboratory for HSP70 screening. Results: 1.The tumor biopsy revealed a strong HSP70 membrane expression and we succeeded to produce a tumor cell line. 2. Two months after surgery 6 sequential cycles of autologous HSP70-activated NK cells reinfusion upon 5 leukapheresis were performed every 4 weeks. After a 3 monthśbreak further 3 reinfusion cycles were performed 3. The absolute number of NK cells in the leukapheresis product ranged between 0.87-1.9 x 10⁹/cycle (16-24% NK cells in PBLs); no significant changes in the total number and subpopulations of lymphocytes were found in the peripheral blood and apheresis product over the 9 cycles. 4. In vitro CD94 cell surface density and cytolytic activity of patient's NK cells against his own HSP70 membrane-positive colon carcinoma cells and K562 was initiated after each cycle upon TKD/IL-2 stimulation. 5. Notably, after the fourth reinfusion cycle unstimulated patient-derived NK cells showed for the first time a cytolytic activity against autologous tumor cells and K562. Despite discontinuation of therapy this in vivo activity persisted for 3 months and than started to decline. Only one single cycle of in vitro TKD/IL-2 stimulation could completely restore the in vivo NK cell activity. 6. Most remarkably, this in vivo NK cell activity was still detectable 1.5 years after completion of our therapy. The patient died of advanced disease 2.5 years after diagnosis of the relapse.Conclusion: This kinetics of initiation and maintenance of an in vivo cytolytic response against HSP70-positive tumors corroborates our data from the phase I clinical trial, showing that irrespective of the tumor entity, stage and previous therapies NK cell activity was reproducibly initiated after 4 reinfusion cycles of TKD/IL-2 activated autologous NK cells. Furthermore, this is the first observation of a long-lasting in vivo cytolytic response of NK cells against HSP70 positive tumors upon TKD/IL-2 stimulation.