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?(Fig.5a).5a). immunofluorescence staining. IL-PLD exhibits more cytotoxicity than PLD in CD22 positive cell lines but does not increase killing of CD22 negative cells. The IC50 of IL-PLD is 3.1 to 5.4 times lower than that of PLD in CD22+ cell lines while the IC50 of IL-PLD is equal to that of PLD in CD22- cells. Furthermore, IL-PLD remained bound to the CD22+ cells after washing and continued to exert cytotoxic effects, while PLD and NL- doxorubicin could easily be washed from these cells. Keywords: HB22.7, CD22, Non-Hodgkins lymphoma, Liposomes, Doxorubicin Introduction NHL are a heterogeneous group of lymphoid malignancies, 80C90% of which are of B-cell origin [1]. NHL is the sixth most common cause of cancer-related deaths in the US, with incidence rates almost doubling since the 1970s [2]. Most NHL are initially responsive to chemotherapy, but relapse is common. Furthermore, the efficacy of chemotherapy is limited by toxicity [1]. One of the most important drugs used in NHL therapy is doxorubicin (DXR). DXR is an anthracycline antibiotic that intercalates into DNA in rapidly dividing cells, thereby inhibiting nucleic acid synthesis [3]. Dose-limiting toxicities of Esomeprazole sodium DXR include cardiomyopathy [4] and myelosuppression [5, 6]. Other toxicities include alopecia, hyperpigmentation of nail beds and dermal folds, nausea, Esomeprazole sodium vomiting, and stomatitis [5]. Pegylated-liposomal formulations (PLD) of DXR (such as Doxil) have prolonged and higher exposures in plasma and tumor, increased antitumor activity in preclinical models, and reduced toxicity [7, 8] compared with non-liposomal (NL) DXR. The increased DXR accumulation in tumors and the decrease in toxicity is thought to be due to the ability of liposomes to extravasate through fenestrated tumor vessels, while in normal tissues, liposomes tend to be confined in the intravascular space since normal vessels are less fenestrated than tumor vessels [9, 10]. However, toxicity of PLD depends on the lipid formulation, with both Esomeprazole sodium fast and slow DXR release from liposomes being least toxic, while intermediate rates of DXR release have varying toxicities [11]. The use of monoclonal antibodies (mAb) conjugated to liposomal drugs (so called immunoliposomes) can further reduce toxicity and increase efficacy by targeting the liposomal drug to the tumor. PLD has been targeted to tumors using anti-CD19 mAbs with success [11, 12]. CD22 is a B-lymphocyte-specific glycoprotein expressed by nearly all mature B-lymphocytes but disappears upon terminal maturation to plasma cells. The two amino-terminal immunoglobulin (Ig) domains of CD22 mediate cell adhesion with sialic-acid bearing ligands. Besides its function as a cell adhesion molecule, CD22 also modulates signal transduction through the B-cell receptor and upon ligation, CD22 becomes internalized [13C15]. Anti-CD22 mAb such as HB22.7, which bind the two amino-terminal Ig domains and specifically block the interaction of CD22 with its ligand, are effective at inducing proliferative responses in primary B-cells and apoptotic responses in neoplastic B-cells [16]. By Esomeprazole sodium contrast, anti-CD22 mAbs that do not block ligand binding have only modest functional effects [16, 17]. As most NHL express CD22, it is a promising target for immunotherapy. Targeting CD22 not only allows for specificity, but also may facilitate intracellular drug delivery based on CD22-mediated internalization. We previously reported the lymphomacidal properties of HB22.7 in nude mice bearing Raji (human B-cell NHL) xenografts [16]. In this study, HB22.7 was conjugated to PLD and tumor cell targeting, cell accumulation of DXR, and cytotoxicity were assessed treatment for 72?h does not accurately reflect conditions that would occur in the blood circulation treatment with 1?h treatment followed by several washes, replacement with fresh treatment-free media, and assessment 71?h later (Fig. ?(Fig.55 and Table ?Table2).2). The cytotoxicity of IL-PLD, PLD, and NL-DXR decreases under washing conditions (Fig. ?(Fig.5b)5b) versus continuous treatment (Fig. ?(Fig.5a).5a). There is a 3-fold increase in the IC50 of IL-PLD under washing conditions compared to continuous treatment, while there is an 11- and 43-fold increase in the IC50 of PLD and NL-DXR, respectively, under washing conditions compared to continuous treatment (Table ?(Table22). Open in a separate window Fig. 4 IL-PLD shows increased cytotoxicity compared to PLD in CD22+ but not CD22- cell lines. CD22+Raji (a, c) or CD22- Jurkat (b, d) were treated with increasing doses of IL-PLD, PLD, or NL-DXR and viability assessed by trypan blue exclusion. Panels c and d include IC50 calculations Table 1 IC50 of cell lines continuously treated with IL-PLD, PLD, and NL- DXR for 72?h circulation. Ramos cells (CD22+) were treated with increasing doses of IL-PLD, PLD, or NL-DXR for either 72?h continuously (a), or for 1?h, followed by three washes and replacement with treatment-free media, and incubated for 71?h (b), then assessed by Esomeprazole sodium Fertirelin Acetate trypan blue exclusion Table 2 IL-PLD growth inhibitory effects on CD22+ cell line are enhanced compared to PLD under washing conditions implications as the blood circulation will act to wash chemotherapy away from their intended targets. The inclusion of HB22.7 on IL-PLD will better resist.