Reduced Effect of Inotuzumab Ozogamicin (CMC544) on P-Glycoprotein Positive Malignant B Cells and Its Restoration by Multidrug Resistance Modifiers.
Authors: Takeshita Akihiro , Shinjo Kaori , Yamakage Nozomi , Ono Takaaki , Hirano Isao , Okinaka Kenji , Matsui Hirotaka , Nakamura Satoki , Shigeno Kazuyuki , Maekawa Masato , Ohnishi Kazunori , Ohno Ryuzo 
Published: April 2020

Abstract
Several new agents have been introduced for the treatment for B cell malignancies (BCM) to overcome resistance to rituximab. Inotuzumab ozogamicin (CMC544), a humanized anti-CD22 mAb conjugated to N-acetyl-g-calicheamicin demethyl hydrazide (NAC-calicheamicin DMH), binds CD22, leading to internalization and delivery of calicheamicin inside the cells. We have been studying gemtuzumab ozogamicin (CMA676), another calicheamicin-conjugated mAb targeting CD33, and we have reported several new findings regarding multi-drug resistance (MDR) and modification of surface antigens. In this study, we attempted to clarify the effect of CMC544 on BCM cells in relation to MDR, and we investigated the restoration effect of the MDR modifier. We also analyzed the effect of CMC544 in relation to CD22 and P-glycoprotein (P-gp) in the samples from BCM.
(Materials and Methods) The cell lines used in this study were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR, respectively. CD22-negative Jurkat, K562 and NB4 cells, and cells obtained from 11 patients with BCM, were also used. CMC544, unconjugated anti-CD22 mAb (G5/44), CMA676, and unconjugated NAC-calicheamicin DMH were kindly provided by Wyeth Pharmaceutical Co., Ltd. The amount of cell surface antigens and P-gp were analyzed by flow cytometry (FCM). For P-gp analysis, cells underwent a reaction with biotinylated MRK16 mAb or with a subclass-matched control mAb and were then stained with streptavidin-Cy-7. P-gp function was determined by intracellular rhodamine-123 (Rh123) accumulation and its enhancement by MDR modifiers, PSC-833 (Novartis) and MS209 (Mitsui), as previously described. The effect of CMC544 was analyzed by cell count, cell viability, and cell cycle distribution on FCM. It was also determined with or without a MDR modifier. Relationships between the CMC544 effect and the amount of P-gp or CD22 were examined statistically.
(Results) A dose-dependent, selective cytotoxic effect of CMC544 was observed in cell lines that expressed CD22. CMC544 is not effective on P-gp-expressing MDR sublines, compared with parental cell lines. MDR modifiers restored the cytotoxic effect of CMC544 in P-gp-expressing sublines. In clinical samples, the cytocidal effect of CMC544, estimated from the fraction of cells in the hypo-diploid portion of the cell cycle, was inversely related to the amount of P-gp estimated by MRK16 mAb (P=0.04), and to the P-gp function assessed by intracellular Rh123 accumulation in the presence of PSC833 or MS209 as MDR modifier (P=0.02 and P=0.01, respectively). Additionally, these MDR modifiers reversed CMC544 resistance in P-gp-expressing CD22-positive cells. On the other hand, the cytotoxic effect of CMC544 was positively correlated with the amount of CD22 (P=0.01).
(Conclusion) This study demonstrates that the CMC544 effect depends on the amounts of P-gp and CD22. We might be able to predict the clinical effects of this drug based on these factors. The treatment of BCM has progressed extremely rapidly, and we can now select particular drugs more specifically among many promising agents. Our results contribute to the development of new strategies to treat BCM. In CD22-positive BCM with P-gp, combined use of CMC544 and MDR modifiers may be more beneficial.