Stefan Zahn

Preclinical characterization of 1-7F9, a novel human anti―KIR receptor therapeutic antibody that augments natural killer―mediated killing of tumor cells

Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3-positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.

Antibody-Mediated Targeting of the Orai1 Calcium Channel Inhibits T Cell Function

Despite the attractiveness of ion channels as therapeutic targets, there are no examples of monoclonal antibodies directed against ion channels in clinical development. Antibody-mediated inhibition of ion channels could offer a directed, specific therapeutic approach. To investigate the potential of inhibiting ion channel function with an antibody, we focused on Orai1, the pore subunit of the calcium channel responsible for store-operated calcium entry (SOCE) in T cells. Effector T cells are key drivers of autoimmune disease pathogenesis and calcium signaling is essential for T cell activation, proliferation, and cytokine production. We show here the generation of a specific anti-human Orai1 monoclonal antibody (mAb) against an extracellular loop of the plasma membrane-spanning protein. The anti-Orai1 mAb binds native Orai1 on lymphocytes and leads to cellular internalization of the channel. As a result, T cell proliferation, and cytokine production is inhibited in vitro. In vivo, anti-Orai1 mAb is efficacious in a human T cell-mediated graft-versus host disease (GvHD) mouse model. This study demonstrates the feasibility of antibody-mediated inhibition of Orai1 function and, more broadly, reveals the possibility of targeting ion channels with biologics for the treatment of autoimmunity and other diseases.

Generation and Characterization of Monoclonal Antibodies against the Transcription Factor Nkx6.1

We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine β-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing β-cells, making this transcription factor a unique β-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/κ subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the β-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies. (J Histochem Cytochem 54:567-574, 2006)