Abstract 3277: Optimized treatment schedule with the new generation CD22-targeting immunotoxin LMB11 induces near MRD-negativity in an ALL mouse model
Authors: Mueller Fabian , Liu XiuFen , Wayne Alan , Pastan Ira 
Published: August 2015

Abstract
Objective: The Pseudomonas exotoxin (PE) based anti-CD22 immunotoxin Moxetumomab pasudotox (HA22) has a response rate of 95% in hairy cell leukemia (HCL) (Kreitman et al 2012), but only about 25% of children with multiply relapsed ALL achieve a complete response (CR) (Wayne et al AACR 2014). HA22 is highly cytotoxic to B-lineage leukemia and lymphoma lines in vitro and in subcutaneous mouse models (Onda et al 2011). HA22 is also active vs primary blasts from 24/35 (68%) children with ALL (Mussai et al 2010). Since there is a significant difference between ALL patient cells in vitro and response rate in patients, we have investigated possible mechanisms of HA22 resistance in mice to aid development of methods to increase response rates in humans.
Methods: We used a modified anti-CD22 immunotoxin, LMB11, which can be safely given to mice at a 10-fold higher dose than HA22 (Bera et al Leuk Lymph 2014). NSG mice were inoculated with 5 million KOPN8 cells I.V. on day 1 and treated on days 8, 10, 12, 14 and 16 with 50 μg LMB11. Bone marrow (BM) samples were analyzed by FLOW for KOPN8 infiltration and apoptosis. Bio-distribution studies used Alexa647-coupled LMB11 and Alexa647 beads to quantify number of LMB11 molecules taken up per cell. Cells were treated with LMB11 for various times, washed, re-plated for 3d, and cell death measured by Annexin V-PE/7-AAD in Flow.
Results: KOPN8 cells are very sensitive to LMB11 (IC50, 0.5 ng/ml) in vitro. Maximal ADP-ribosylation of EF2 occurs at 6h, protein synthesis stops at 8h, MCL1 levels fall at 8h, and PARP cleavage begins at 10h. Untreated KOPN8-inoculated NSG mice survived 21.5 d. When treated with 50ug x 5 of LMB11, mice survived 30 d. There was no significant apoptosis of KOPN8 cells in murine BM during LMB11 dosing. ALL BM infiltration remained at 5% during dosing. Four days after the last dose, BM infiltration increased rapidly. Cellular uptake of LMB11 was 200-fold over the amount required to kill cells in vitro.
To identify potential resistance mechanisms, we treated KOPN8 for various times in vitro and found that KOPN8 needed a 9h exposure to LMB11 for &gt;95% of cells to die. There was p38 MAPK activation at 6h, which is probably responsible for cell cycle arrest of KOPN8 cells in the BM.
Because LMB11 has a short half-life (30 min) in mice and because long exposure time is needed to induce apoptosis in vitro, we treated with 4 doses of 25 ug LMB11 every 3h for 3 days to maintain adequate blood levels. This resulted in near MRD-negative BM (0.004%) by flow.
We screened many drugs for their ability to shorten the exposure time needed to induce &gt;95% cell death by LMB11 and found that H89, nilotinib, imatinib and verapamil were active.
Conclusion: KOPN8 cells need prolonged immunotoxin exposure to drive cells into apoptosis. HA22 given more frequently or combined with sensitizing agents might increase CR rates in patients with ALL.
Citation Format: Fabian Mueller, XiuFen Liu, Alan Wayne, Ira Pastan. Optimized treatment schedule with the new generation CD22-targeting immunotoxin LMB11 induces near MRD-negativity in an ALL mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3277. doi:10.1158/1538-7445.AM2015-3277