The selection of variable regions affects effector mechanisms of IgA antibodies against CD20.

Blockade of the CD47-SIRPα axis improves lymphoma cell killing by myeloid effector cells, which is an important effector mechanism for CD20 antibodies in vivo. The approved CD20 antibodies rituximab, ofatumumab, and obinutuzumab are of human immunoglobulin G1 (IgG1) isotype. We investigated the impact of the variable regions of these 3 CD20 antibodies when expressed as human IgA2 isotype variants. All 3 IgA2 antibodies mediated antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cellular cytotoxicity (ADCC) by polymorphonuclear cells. Both effector mechanisms were significantly enhanced in the presence of a CD47-blocking antibody or by glutaminyl cyclase inhibition to interfere with CD47-SIRPα interactions. Interestingly, an IgA2 variant of obinutuzumab (OBI-IgA2) was consistently more potent than an IgA2 variant of rituximab (RTX-IgA2) or an IgA2 variant of ofatumumab (OFA-IgA2) in triggering ADCC. Furthermore, we observed more effective direct tumor cell killing by OBI-IgA2 compared with RTX-IgA2 and OFA-IgA2, which was caspase independent and required a functional cytoskeleton. IgA2 variants of all 3 antibodies triggered complement-dependent cytotoxicity, with OBI-IgA2 being less effective than RTX-IgA2 and OFA-IgA2. When we investigated the therapeutic efficacy of the CD20 IgA2 antibodies in different in vivo models, OBI-IgA2 was therapeutically more effective than RTX-IgA2 or OFA-IgA2. In vivo efficacy required the presence of a functional IgA receptor on effector cells and was independent of complement activation or direct lymphoma cell killing. These data characterize the functional activities of human IgA2 antibodies against CD20, which were affected by the selection of the respective variable regions. OBI-IgA2 proved particularly effective in vitro and in vivo, which may be relevant in the context of CD47-SIRPα blockade.

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing.

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these "off-the-shelf" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.