Images were analyzed with Living Image 3.0 software (Caliper Rabbit polyclonal to CNTFR LifeScience). To treat established tumors, antibody therapy was started 5 or 12 days after tumor challenge, and 200?g isoAb or J22. 9\xi per injection administered twice weekly for a period of 6 weeks. 2.8. monoclonal antibody directed against the B cell maturation antigen. The antibody binds to the ligand\binding site of BCMA and blocks APRIL and BAFF. The BCMA\antibody depletes multiple myeloma cells in?vitro through ADCC and CDC. The BCMA\antibody prevents tumor growth in mouse models of multiple myeloma. 1.?Introduction Multiple myeloma (MM) is an aggressive neoplasm resulting from the malignant transformation of plasma cells (PCs) and their precursors (Palumbo and Anderson, 2011; Raab et?al., 2009). High dose chemotherapy followed by autologous stem cell transplantation has prolonged survival after ortho-iodoHoechst 33258 diagnosis by approximately 4C5 years (Hideshima and Anderson, 2002; Munshi and Anderson, 2013; Palumbo and Anderson, 2011; Raab et?al., 2009; Richardson et?al., 2003; Yang and Yi, 2011). The recent introduction of anti\angiogenic drugs like thalidomide or lenalidomide and the proteasome inhibitor bortezomib into the clinic has improved the median survival of MM patients to 5C7 years (Hideshima and Anderson, 2002; Munshi and Anderson, 2013; Palumbo and Anderson, 2011; Raab et?al., 2009; Richardson et?al., 2003; Yang and ortho-iodoHoechst 33258 Yi, 2011). However, despite these advances, MM is still incurable in most patients. Therefore, the need for new drugs for efficient clearance of the malignant cells remains. During the past two decades monoclonal antibodies have increasingly been used for the treatment of hematological malignancies. For example, treatment with Rituximab, a monoclonal antibody (mAb) against CD20, in combination with chemotherapy, has dramatically improved the long\term survival of patients suffering from Non\Hodgkin’s Lymphoma (Cheson and Leonard, 2008). Accordingly, mAbs targeting cell surface molecules expressed on MM cells like CD38, CD70 or CD138 are currently in preclinical development or in early phase clinical trials. Strategies interfering with the tumor growth\promoting bone marrow (BM) environment by targeting B cell growth factors such as IL\6, APRIL, and/or BAFF have also reached the clinic (Munshi and Anderson, 2013; Rossi et?al., 2009; Tai and Anderson, 2011; Yang and Yi, 2011). However, there are as yet no approved antibody\based therapies for MM. In addition, the epitopes of mAbs in clinical development are not exclusively present on MM cells. CD38, for instance, is also expressed on activated B and T cells (Malavasi et?al., 2008) and CD138 is present on epithelial cells (Inki and Jalkanen, 1996). Therefore, we generated an antibody against the B cell maturation antigen (BCMA), which is almost exclusively ortho-iodoHoechst 33258 expressed on plasma blasts and plasma cells but is absent from naive, germinal center and memory B cells (Benson et?al., 2008; Darce et?al., 2007; Good et?al., 2009). BCMA is a receptor for APRIL and BAFF and is known to be important for the survival of long\lived PCs in the BM (O’Connor et?al., 2004). Previous findings by Ryan et?al. (2007) showed that antibodies and antibody\drug conjugates (ADC) directed against BCMA blocked APRIL binding and led to an efficient depletion of MM cells (O’Connor et?al., 2004). It was not shown, however, whether such antibodies could target MM cells and and substantially increases tumor\free survival in a mouse model of MM. Our high resolution structure of the Fab in complex with the extracellular domain of human BCMA provides a detailed picture of the antibody’s epitope and will help to facilitate humanization and sequence optimization. 2.?Methods 2.1. BCMA expression and purification Human BCMA (residues 1C54) was expressed from the pGEX6p\1 vector (GE Healthcare) as ortho-iodoHoechst 33258 an N\terminal glutathione\S\transferase (GST) fusion followed by a PreScission cleavage site. Proteins were expressed in host strain Rosetta2\BL21\DE3, and bacteria were cultured in TB medium at 37?C to an OD600 of 0.5 followed by induction with 60?M Isopropyl \d\1\thiogalactopyranoside (IPTG) and temperature shift to 18?C for overnight expression. Cells were resuspended in buffer A (50?mM HEPES/NaOH, pH 7.5, 500?mM NaCl 1?M DNase (Roche), 500?M Pefabloc (Roth)) and disrupted in a microfluidizer (Microfluidics). Cleared lysates (95,000?g, 1?h, 4?C) were incubated with Benzonase (Novagen) for 30?min at 4?C prior to application to a GSH column (Clontech). Protein was eluted with buffer.