Next, the membrane was washed three times with PBS-T (phosphate buffered saline with Tween 20) and incubated with donkey anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680RD secondary antibodies (LI-COR, USA) for 1 h at RT

Next, the membrane was washed three times with PBS-T (phosphate buffered saline with Tween 20) and incubated with donkey anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680RD secondary antibodies (LI-COR, USA) for 1 h at RT. flow cytometry and cellular binding assays were performed to test the binding ability of labeled or conjugated daratumumab with CD38 biodistribution studies showed similar trends with imaging results. Immunofluorescent staining of tumor tissues verified higher CD38 expression of Ramos than that of HBL-1. Conclusions The role of 89Zr-Df-daratumumab was investigated for evaluating CD38 expression in lymphoma models noninvasively and was found to be to a promising imaging agent of CD38 positive hematological diseases such as MM in future clinical applications. and would, therefore, be useful for accurate diagnosis, prognosis, and therapeutic evaluation. CD38 is usually a 45-kD type II transmembrane glycoprotein that functions in the enzymatic activity of intracellular calcium signaling, receptor-mediated regulation of lymphocyte cell adhesion and signal transduction [5, 6]. CD38 is usually highly expressed on plasma cells, especially on multiple myeloma (MM) cells. However, CD38 is expressed at relatively low levels on most resting natural killer (NK) cells, monocytes, and other various hematological cell types [7]. This suggests that CD38 is a suitable biomarker for MM [8]. Moreover, CD38 has been found to be an adverse prognostic marker in chronic lymphocytic leukemia [9]. Therefore, evaluation of CD38 expression could provide useful information about the treatment and prognosis of CD38-positive hematologic malignancies. Daratumumab (Darzalex; Janssen Biotech, Inc) is the first anti-CD38 human immunoglobulin G1 kappa (IgG1-) mAb [7, 10]. It was developed from the immunization of human immunoglobulin transgenic CB-6644 mice with recombinant CB-6644 CD38 protein [11]. Daratumumab binds to CD38 and induces the death of CD38-expressing cells via diverse cytotoxic mechanisms that include complement-dependent cytotoxicity, antibody-dependent cytotoxicity, antibody-dependent cellular phagocytosis, apoptosis, and the inhibition of enzymatic activity of CD38 [11C13]. Daratumumab exhibited therapeutic effects even at low concentrations in a SCID mouse xenograft tumor model [13]. Moreover, daratumumab not only showed inhibitory on MM tumor models but also on B cell tumor models [11], suggesting a potential application in lymphoma. In our previous study, 89Zr-labeled daratumumab was developed as a PET probe to visualize CD38 expression in lung cancer models [14]. However, the relatively low tumor uptake (highest at 8.1 1.2 %ID/g) limited its potential for lung tumor imaging. To date, there have been no reports about CD38 PET imaging in lymphoma. Herein, we prepare 89Zr-labeled daratumumab for imaging of CD38 expression in different B-cell lymphoma models. We hypothesized that PET imaging using 89Zr-Df-daratumumab would allow for differentiation of CD38 expression and sensitive detection of CD38-positive lymphoma, and therefore for the guidance of clinical treatment. Materials and methods Conjugation and radiolabeling The CB-6644 preparation of 89Zr (t1/2 = 78.4 h) radiolabeled daratumumab was performed through conjugation with SCN-Bn-deferoxamine (Df), as described in previous protocols [14, 15]. Briefly, 4 mg of daratumumab was added to 500 L of PBS and adjusted to a pH of 8.5 C 9.0 with Na2CO3 (0.1 M), then Df dissolved in dimethyl sulfoxide was added at a 1:10 molar ratio. The reaction was kept at room heat (RT) for 2 h. Df-daratumumab was then purified using PD-10 columns (GE Healthcare) with PBS as the mobile phase. For 89Zr labeling, 148 MBq (4 mCi) of 89Zr-oxalate were produced using a PETrace cyclotron (GE Healthcare) and diluted in 0.5 mL of HEPES buffer (0.5 M) at the adjusted pH of 7.0 CD350 using Na2CO3 (2M). 600 g (150 g/mCi) of Df-daratumumab was added and incubated for 1h at 37 C. The 89Zr-Df-daratumumab product was then purified using PD-10 columns and measured for its radiochemical yields. 89Zr labeled human serum IgG was used as a non-specific control tracer and prepared with similar methods as mentioned above. Cell culture Human B lymphocyte cell lines Ramos, Daudi, Raji (Burkitts lymphoma), Rec-1 (Mantle cell lymphoma), and HBL-1 (Human diffuse large B-cell lymphoma) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Invitrogen) and CB-6644 1% penicillin/streptomycin (Hyclone) at 37 C in a humidified incubator with 5% CO2. Western blot For Western blot, lymphoma cells were centrifuged and harvested. Total protein concentration of each cell line was measured using a Pierce Coomassie protein assay kit (ThermoFisher Scientific). 40 g of total protein was loaded into a 4 C 12% Bolt Bis-Tris Plus gel (ThermoFisher Scientific), along with Chameleon Duo ladder protein marker (LI-COR Biosciences, USA). Electrophoresis was performed at 110 mV and 4 C for 75 min. All proteins were then transferred to a nitrocellulose membrane using an iBlot 2 (ThermoFisher Scientific) for 7 min. The membrane was blocked with Odyssey blocking buffer (LI-COR, USA) for 1 h at RT and further incubated with a mouse anti-human CD38 antibody (1:1000) and rabbit anti-human -tubulin antibody (1:2000) overnight at 4 C. All primary antibodies were purchased.