UNLABELLED: Our aim was to construct and characterize (111)In-nuclear translocation sequence (NLS)-7G3, an Auger electron-emitting radioimmunotherapeutic agent that preferentially recognizes the expression of CD123 (interleukin-3 receptor [IL-3R] α-subchain) in the absence of CD131 (IL-3R β-subchain) displayed by leukemia stem cells. METHODS: Monoclonal antibody 7G3 was modified with 13-mer peptides [CGYGPKKKRKVGG] harboring the NLS of SV-40 large T-antigen and with diethylenetriaminepentaacetic acid for labeling with (111)In. Immunoreactivity was evaluated in a competition radioligand binding assay and by flow cytometry. Nuclear localization of (111)In-NLS-7G3 was studied by cell fractionation in CD123(+)/CD131(-) acute myelogenous leukemia (AML)-3, -4, and -5 cells or in primary AML or normal leukocytes. Micro-SPECT was performed in nonobese diabetic (NOD)/severe combined immune deficient (SCID) mice engrafted subcutaneously with Raji-CD123 tumors or with disseminated AML-3 or -5 cells. The cytotoxicity of (111)In-NLS-7G3 on AML-5 cells was studied after 7 d in culture by trypan blue dye exclusion. DNA damage was assessed using the γ-H2AX assay. RESULTS: NLS-7G3 exhibited preserved CD123 immunoreactivity (affinity, 4.6 nmol/L). Nuclear importation of (111)In-NLS-7G3 in AML-3, -4, or -5 cells was specific and significantly higher than unmodified (111)In-7G3 and was greater in primary AML cells than in normal leukocytes. Rapid elimination of (111)In-NLS-7G3 in NOD/SCID mice prevented imaging of subcutaneous Raji-CD123 tumors. This phenomenon was Fc-dependent and IgG(2a) isotype-specific and was overcome by the preadministration of excess IgG(2a) or using (111)In-NLS-7G3 F(ab')(2) fragments. AML-3 and -5 cells were engrafted into the bone marrow or spleen or at extramedullary sites in NOD/SCID mice. Micro-SPECT/CT with (111)In-NLS-7G3 F(ab')(2) showed splenic involvement, whereas foci of disease were seen in the spine or femur or at extramedullary sites in the brain and lymph nodes using (111)In-NLS-7G3 IgG(2a). The viability of AML-5 cells was reduced by exposure in vitro to (111)In-NLS-7G3; this reduction was associated with an increase in unrepaired DNA double-strand breaks. CONCLUSION: (111)In-NLS-7G3 is a promising novel Auger electron-emitting radioimmunotherapeutic agent for AML aimed at the leukemia stem cell population. Micro-SPECT/CT was useful for visualizing the engraftment of leukemia in NOD/SCID mice.
UNLABELLED: Our aim was to construct and characterize (111)In-nuclear translocation sequence (NLS)-7G3, an Auger electron-emitting radioimmunotherapeutic agent that preferentially recognizes the expression of CD123 (interleukin-3 receptor [IL-3R] α-subchain) in the absence of CD131 (IL-3R β-subchain) displayed by leukemia stem cells. METHODS: Monoclonal antibody 7G3 was modified with 13-mer peptides [CGYGPKKKRKVGG] harboring the NLS of SV-40 large T-antigen and with diethylenetriaminepentaacetic acid for labeling with (111)In. Immunoreactivity was evaluated in a competition radioligand binding assay and by flow cytometry. Nuclear localization of (111)In-NLS-7G3 was studied by cell fractionation in CD123(+)/CD131(-) acute myelogenous leukemia (AML)-3, -4, and -5 cells or in primary AML or normal leukocytes. Micro-SPECT was performed in nonobese diabetic (NOD)/severe combined immune deficient (SCID) mice engrafted subcutaneously with Raji-CD123tumors or with disseminated AML-3 or -5 cells. The cytotoxicity of (111)In-NLS-7G3 on AML-5 cells was studied after 7 d in culture by trypan blue dye exclusion. DNA damage was assessed using the γ-H2AX assay. RESULTS: NLS-7G3 exhibited preserved CD123 immunoreactivity (affinity, 4.6 nmol/L). Nuclear importation of (111)In-NLS-7G3 in AML-3, -4, or -5 cells was specific and significantly higher than unmodified (111)In-7G3 and was greater in primary AML cells than in normal leukocytes. Rapid elimination of (111)In-NLS-7G3 in NOD/SCIDmice prevented imaging of subcutaneous Raji-CD123tumors. This phenomenon was Fc-dependent and IgG(2a) isotype-specific and was overcome by the preadministration of excess IgG(2a) or using (111)In-NLS-7G3 F(ab')(2) fragments. AML-3 and -5 cells were engrafted into the bone marrow or spleen or at extramedullary sites in NOD/SCIDmice. Micro-SPECT/CT with (111)In-NLS-7G3 F(ab')(2) showed splenic involvement, whereas foci of disease were seen in the spine or femur or at extramedullary sites in the brain and lymph nodes using (111)In-NLS-7G3 IgG(2a). The viability of AML-5 cells was reduced by exposure in vitro to (111)In-NLS-7G3; this reduction was associated with an increase in unrepaired DNA double-strand breaks. CONCLUSION: (111)In-NLS-7G3 is a promising novel Auger electron-emitting radioimmunotherapeutic agent for AML aimed at the leukemia stem cell population. Micro-SPECT/CT was useful for visualizing the engraftment of leukemia in NOD/SCIDmice.
Authors: Arthur E Frankel; Jung H Woo; Chul Ahn; Naveen Pemmaraju; Bruno C Medeiros; Hetty E Carraway; Olga Frankfurt; Stephen J Forman; Xuezhong A Yang; Marina Konopleva; Francine Garnache-Ottou; Fanny Angelot-Delettre; Christopher Brooks; Michael Szarek; Eric Rowinsky Journal: Blood Date: 2014-05-23 Impact factor: 22.113
Authors: Simon Beaudoin; Michel Paquette; Laurent Fafard-Couture; Mylene A Tremblay; Roger Lecomte; Brigitte Guérin; Jeffrey V Leyton Journal: J Vis Exp Date: 2018-03-08 Impact factor: 1.355
Authors: Rashad Al-Salahi; Moustapha E Moustapha; Hatem A Abuelizz; Abdulrahman I Alharthi; Khalid A Alburikan; Ismail T Ibrahim; Mohamed Marzouk; Mohamed A Motaleb Journal: Saudi Pharm J Date: 2018-06-06 Impact factor: 4.330