Enhanced Killing of Human B Lymphoma Targets by Combined Use of Cytokine Induced Killer (CIK) Cultures and Anti-CD20 Antibodies
Authors: Pievani Alice , Belussi Camilla , Klein Christian , Rambaldi Alessandro , Golay Josée , Introna Martino 
Published: October 2019

Abstract
Abstract 4285
Cytokine induced killer (CIK) cells are immune-effector cells that can be expanded in vitro in presence of rhIL-2, starting from peripheral blood mononuclear cells stimulated by interferon-γ and anti-CD3 antibody. CIK cultures at the end of in vitro expansion contain a mean of 40–75% CD3+CD56+ CIK cells, 20–60% CD3+CD56- T cells and 1–10% CD3-CD56+ NK cells. They show MHC-unrestricted cytotoxicity towards neoplastic but not normal targets. Their ease of production in vitro and anti-tumor potential have made them suitable candidates for cell therapy programs in solid and hematopoietic tumour treatment. CIK cells have shown cytotoxic activity in vitro against hematopoietic neoplastic cells, including B Non-Hodgkin's lymphoma (B-NHL). Other biological treatments available for B-NHL are the anti-CD20 antibodies such as type I Rituximab and a new generation glycoengineered type II GA101 antibody. These antibodies are thought to act mostly through immune mediated mechanisms including phagocytosis, complement mediated cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). GA101 has reduced CDC activity compared to type I anti-CD20 antibodies such as Rituximab. In addition, GA101 was defucosylated in its Fc portion to mediate increased ADCC. We have investigated the possibility of combining adoptive immunotherapy by Cytokine Induced Killer (CIK) cells with anti-CD20 type I Rituximab and type II GA101mAb, to optimize B-NHL therapy. CIK cultures alone demonstrated significant cytotoxic activity against a panel of B-NHL cell lines or freshly isolated samples, in either an autologous or allogeneic combination (26-27% killing at 30:1 ET ratio). This natural cytotoxic activity was mainly due to the predominating CD3+CD56+ CIK population (40-75%) present in the cultures. The addition of anti-CD20 mAbs increased CIK mediated cytotoxicity versus B lymphoma target cells and major enhancement was observed with GA101 compared to Rituximab (respectively 34% versus 16% increased lysis at 10:1 E:T ratio). This enhancement was mainly due to ADCC mediated by the small NK cell fraction (1-10%) present in CIK cultures because NK depletion by CD5 immunoselection at the end of expansion did not abolish the basal natural cytotoxicity of CIK cultures but abolished the enhancement observed in presence of anti-CD20 antibodies. The activation of NK cells in CIK cultures, evaluated by CD107a mobilization, was much more effective using GA101 rather than Rituximab (respectively 28% versus 19% CD107a+, p<0.005). Furthermore, in presence of human serum, Rituximab-mediated NK cell activation was inhibited to 70%, whereas the GA101-mediated was fully maintained. This inhibition was presumably due to complement C3 deposition, since it was not observed with either heat inactivated serum or high concentrations of human immunoglobulins. Inhibition was however observed with serum plus anti-C5 antibody eculizumab, which blocks the complement cascade downstream from C3. Lack of inhibition by serum of GA101-mediated NK cell degranulation was probably due to the higher affinity binding of this mAb to CD16 and not to a lower C3 activation, because at high concentrations, GA101 is nearly as efficient as Rituximab at activating complement and C3 deposition. Finally, addition of serum to CIK and mAbs did not modify overall the target cell killing by either antibody. More importantly, the use of a partially complement and CIK resistant lymphoma cell line such as WSU-NHL showed that resistance could be reverted by combined exposure to CIK cultures and monoclonal anti-CD20 antibodies. Indeed overall lysis of WSU-NHL, even in presence of serum, was significantly increasedby both anti-CD20 antibodies, and most effectively by GA101, compared to CIK cells alone. This was due to the combined action of CDC, ADCC and CIK mediated natural killing. In conclusion, CIK cultures could be used to treat B-NHL patients in both an autologous or allogeneic setting. Furthermore Rituximab but even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL. These data may open the way to possible therapeutic exploitation of combined strategies of cell mediated and antibody mediated immunotherapy protocols which make use of different mechanisms of action to try and overcome resistance.

Disclosures:
Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Rambaldi:Roche: Honoraria. Golay:Roche: Honoraria. Introna:Roche: Honoraria.