SummaryThe effect of CMC-544, a calicheamicin-conjugated anti-CD22 monoclonal antibody, was analysed in relation to CD22 and P-glycoprotein (P-gp) in B-cell chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) in vitro. The cell lines used were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR. Cells obtained from 19 patients with B-cell CLL or NHL were also used. The effect of CMC-544 was analysed by viable cell count, morphology, annexin-V staining, and cell cycle distribution. A dose-dependent, selective cytotoxic effect of CMC-544 was observed in cell lines that expressed CD22. CMC-544 was not effective on Daudi/MDR and Raji/MDR cells compared with their parental cells. The MDR modifiers, PSC833 and MS209, restored the cytotoxic effect of CMC-544 in P-gp-expressing sublines. In clinical samples, the cytotoxic effect of CMC-544 was inversely related to the amount of P-gp (P = 0AE003), and to intracellular rhodamine-123 accumulation (P < 0AE001). On the other hand, the effect positively correlated with the amount of CD22 (P = 0AE010). The effect of CMC-544 depends on the levels of CD22 and P-gp. Our findings will help to predict the clinical effectiveness of this drug on these B-cell malignancies, suggesting a beneficial effect with combined use of CMC-544 and MDR modifiers.
We studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (Po0.001 in both cells) after incubation with CMC-544, but CD20 level remained constant or increased for 12 h. Similar results were obtained in cells from 12 patients with BCM. The antiproliferative and apoptotic effect of CMC-544 were greater than that of rituximab. The ADCC of rituximab was not enhanced by CMC-544. Thus, the combination of CMC-544 and rituximab increased the in vitro cytotoxic effect in BCM cells, and sequential administration for 12 h proceeded by CMC-544 was more effective. The reduction of CD55 and the preservation of CD20 after incubation with CMC-544 support the rationale for the combined use of CMC-544 and rituximab.
Rituximab has greatly improved the prognosis of B cell malignancies (BCM). However, several resistance mechanisms have been reported, including the inhibition of apoptosis, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) as well as modulation of CD20 antigen and increased complement-related cytotoxicity inhibition factor (CD55). The calicheamicin-conjugated anti-CD22 monoclonal antibody (mAb), CMC544, is one of the new promising agents, and effective on several types of BCM in vitro, especially when used in combination with rituximab. However, details regarding the cytotoxic effects of CMC544 and mechanisms triggering cell death, particularly when it is combined with rituximab, have not been elucidated. We studied cell cycle features of CMC544 on BCM used alone or in combination with rituximab, and analyzed quantitative alteration of target molecules such as CD20, CD22 and CD55.(Materials and Methods) The cell lines used in this study were CD22-positive Daudi and Raji cells, and CD22-negative Jurkat and NB4 cells. Cells obtained from 8 patients with BCM were also used. CMC544, unconjugated anti-CD22 mAb (G5/44), and free NAC-calicheamicin DMH were provided by Wyeth Pharmaceutical Co., Ltd. The effects of CMC544 were analyzed by incubating the cells at the concentrations of 0–100μg/ml for 0–96h, then by measuring cell count, cell viability, 3H-thymidine incorporation and cell cycle distribution on flow cytometry (FCM) as well as by video microscopic observation. The amounts of CD20, CD22 and CD55 antigens were analyzed by FCM, laser scanning microscope. The process of CMC544-induced cell death was assessed by Apocyto®. Three possible mechanisms of the combined effect of CMC544 and rituximab were investigated separately by direct effect of the mAb, CDC and ADCC. (Results) Analysis of the cell cycle distribution indicated that CD22-positive cells were temporally arrested at the G2/M phase after 6–12h incubation with CMC544, and the hypodiploid proportion increased after 24–72h. CD22-positive cells enlarged after 12–24h incubation with CMC544. Apoptotic morphological changes such as bleb formation and cell shrinkage were observed in 35–57% of the cells. These changes were not observed after incubation with G5/44. Apocyto® stain showed a continuous pattern from early to late apoptosis. Similar effects were observed in clinical samples that expressed CD22. The amounts of CD22 and CD55 were significantly reduced 3h after incubation with CMC544, while the CD20 expression was maintained 24h after incubation with CMC544. Incubation of the cells for 12–24h with CMC544 and then for 12hr with rituximab induced 1.2–2.1 times more anti-proliferation, apoptosis, CDC and ADCC than simultaneous incubation.(Conclusion) The effect of CMC544 was clearly observed as G2/M arrest following increase of the hypodiploid proportion in the cell cycle distribution, and this was a useful method to investigate the effect of CMC544. Combination of CMC544 and rituximab enhanced the cytotoxic effect, and sequential administration was more effective. The reduction of CD55 and continued expression of CD20 after the incubation with CMC544 supported the above observation. Further investigations are required to find the most effective treatment regimen to overcome drug resistance of B cell malignancies.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.