Leukemia (2007) 21, 1405–1412 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE A CD19-specific single-chain immunotoxin mediates potent apoptosis of B-lineage leukemic cells M Schwemmlein1, J Stieglmaier1, C Kellner1, M Peipp2, D Saul1, F Oduncu3, B Emmerich3, B Stockmeyer4, P Lang5, JD Beck6 and GH Fey1 1Chair of Genetics, University of Erlangen-Nuremberg, Erwin-Rommel-Strasse 3, Erlangen, Germany; 2Division of Stem Cell Transplantation and Immunotherapy, 2nd Medical Department, University of Kiel, Schittenhelmstrasse 12, Kiel, Germany; 3Medizinische Klinik Innenstadt, Klinik der Ludwig-Maximilians-Universitaet, Ziemssenstrasse 1, Munich, Germany; 4Division of Hematology, Medical Department 3, University of Erlangen-Nuremberg, Krankenhausstrasse 12, Erlangen, Germany; 5Department of Pediatric Oncology, University Children’s Hospital, University of Tuebingen, Hoppe-Seyler-Strasse 1, Tuebingen, Germany and 6Department of Pediatric Oncology, University Children’s Hospital, University of Erlangen-Nuremberg, Loschgestrasse 15, Erlangen, Germany CD19 is a B-lineage-specific transmembrane signaling protein leukemias (ALL), common ALL, non-Hodgkin-lymphomas participating in the control of proliferation and differentiation. It (NHL), chronic B-lymphocytic leukemia (B-CLL) and hairy-cell is present at high surface density on chronic B-lymphocytic leukemias (HCL3). The antigen is not shed from the surface of leukemia (B-CLL) cells and cells of other B-cell malignancies, malignant cells and is internalized after antibody binding.4 and is a prime target for therapy with antibody-derived agents. Many attempts have been made to target malignant cells via Owing to these favorable properties, CD19 appears to be an CD19, but to date none of these agents have received drug attractive target for the treatment of B-cell malignancies with approval. Here we report the design of a monovalent immuno- antibody-derived therapeutics. Consequently, whole unconju- toxin consisting of a CD19-specific single-chain Fv antibody gated CD19 monoclonal antibodies (mAbs) have been used in fragment fused to a derivative of Pseudomonas Exotoxin A. preclinical and clinical studies,5–8 but to date have not achieved This fusion protein induced efficient antigen-restricted apopto- convincing clinical results. Their interaction with Fc-receptors sis of several human leukemia- and lymphoma-derived cell 9,10 lines including Nalm-6, which it eliminated at an effective has been improved through glyco-engineering, and further concentration (EC50) of 2.5 nM. The agent displayed synergistic improvements through the use of fully human mAbs may still toxicity when used in combination with valproic acid and lead to the development of successful intact therapeutic CD19 cyclosporin A in cell-culture assays. It induced apoptosis of antibodies in the future. primary malignant cells in 12/12 samples from B-CLL patients, The clinically used CD20 mAb Rituximab owes its success including patients responding poorly to fludarabine, and of largely to effector mechanisms such as antibody-dependent cells from one pediatric acute lymphoblastic leukemia patient. In NOD/SCID mice transplanted with Nalm-6 cells, the toxin cellular cytotoxicity (ADCC) and the activation of comple- 11 prevented engraftment and significantly prolonged survival of ment. However, interactions of therapeutic antibodies with treated mice. Owing to its efficient antigen-restricted antileu- Fc-receptors on a variety of effector cells can also cause kemic activity, the agent deserves further development towards detrimental effects. Bispecific antibodies targeting CD19 and clinical testing. CD3 showed potent effects against tumor cells in cell-culture Leukemia (2007) 21, 1405–1412; doi:10.1038/sj.leu.2404687; assays but did not convince in clinical trials,12,13 probably in published online 10 May 2007 Keywords: CD19; immunotoxin; exotoxin A; antibody therapy; part because they were absorbed by Fc-receptors on other cells 14 leukemia than the intended effector cells. To reduce these undesired effects, recombinant bispecific antibody constructs have been produced, consisting of single-chain Fv antibody fragments (scFvs) specific for CD19 fused to scFvs directed against trigger molecules on effector cells. Typical trigger molecules used were Introduction CD3 on T lymphocytes and CD16 on natural killer (NK) cells. These molecules deliberately lacking Fc-portions were success- CD19 is a transmembrane glycoprotein of the immunoglobulin ful in cell-culture studies, and some are under current clinical superfamily, which participates in the communication of B- investigation.15–18 lymphoid cells with their environment and the control of To avoid the problems of CD19-specific agents relying on proliferation and differentiation in response to external sig- effector cells, we aimed here for an agent employing an effector- 1,2 nals. The protein is broadly expressed through most stages of cell-independent mechanism of action. In certain therapeutic B-cell maturation from early pro-B-cells onwards and is down- settings, effector cells may not be present in sufficient quality regulated on plasma cells. It is not expressed outside the and numbers. There is a clearly stated need for CD19-specific B-cell lineage. Many B-lymphoid malignancies display this agents, for example in the treatment of pediatric leukemias. At antigen, including most pro- and pre-B-cell acute lymphoblastic the end of a conditioning chemotherapy, the blast titer often is still not reduced far enough to provide a reasonable prospect for Correspondence: Professor GH Fey, Chair of Genetics, University of a successful outcome of a stem-cell transplantation.19 In this Erlangen-Nuremberg, Erwin-Rommel-Strasse 3, D-91058 Erlangen, situation, an effector-cell-independent agent such as an im- Germany. E-mail: [email protected] munotoxin is desirable to further reduce the blast cell titer, Received 8 January 2007; revised 6 March 2007; accepted 7 March because the effector cells have also been damaged by the 2007; published online 10 May 2007 conditioning regime. CD19 single-chain Fv immunotoxin M Schwemmlein et al 1406 Various immunotoxins specific for CD19 have been generated Mice and tested for antileukemic effects. In the majority of cases, Female NOD/SCID mice (M&B, Ry, Denmark) were maintained plant-derived toxins such as ricin, saporin and derivatives were under sterile and standardized environmental conditions used.20–22 In clinical trials, these agents have not produced (22711C room temperature, 50710% relative humidity, 12 h durable responses and often gave rise to dose-limiting toxicities, light–dark cycle) and received autoclaved food and bedding mostly in form of vascular leak syndrome (VLS22,23). In addition, (ssniff Spezialdia¨ten, Soest, Germany) and acidified (pH 4.0) neutralizing antibodies against the immunoconjugates were drinking water ad libitum. All experiments were performed observed due to their murine origin and the plant-derived toxin according to the German Animal Protection Law with permis- components. Mutated variants of these toxins with greatly sion from the responsible local authorities. reduced binding to endothelial cells have been created, which caused fewer VLS symptoms in murine studies.24 Another series of immunotoxins have been produced employing bacterial Construction and expression of CD19-Exotoxin A’ toxins such as a truncated version of Pseudomonas Exotoxin A fusion protein (CD19-ETA0) (ETA0) as the effector component. In these fusion proteins, the The sequence coding for the CD19-specific scFv was excised toxin component was fused to antibody fragments specific for a from a previously cloned pAK100 vector construct34 and cloned number of tumor cell target antigens.25–29 Among the most into the expression vektor pet27b( þ ) (Novagen, Inc.) as advanced representatives of this generation is BL22, a CD22 previously described,29 resulting in the expression vector antibody fragment coupled to ETA0. In clinical trials for the pet27b( þ )-STREP-His-CD19-ETA-KDEL. The CD19-ETA0 fusion treatment of HCL, this agent showed convincing effects, with protein was expressed under osmotic stress conditions.29 only about 25% of the patients developing neutralizing antibodies.30 Here we have chosen to design an scFv-ETA0 fusion protein, because: (a) the absence of an Fc-portion offered Flow cytometric analysis the possibility of reduced interactions with Fc-receptors on Binding of CD19-ETA0 to cells was analyzed using a FACSCa- undesired types of effector cells; (b) scFvs should have reduced libur FACS instrument and CellQuest software (Becton Dick- immunogenicity, because most human-anti-mouse antibody inson, Mountain View, CA, USA). Cells (2.5 Â 105) were responses (HAMA) are directed against the Fc-portion of whole incubated for 30 min on ice with 20 ml of the immunotoxin at antibodies;31 and (c) ETA0 toxins have been reported to show an a concentration of 5 mg/ml. A non-related CD33-ETA0 immuno- approximately 1000-fold lower affinity for endothelial cells than toxin29 served as control for background staining. The cells were ricin-derived toxins,32 and should therefore cause far fewer VLS washed with PBA (phosphate-buffered saline (PBS) containing symptoms. Finally, the clinical trials with BL22 have shown only 0.1% bovine serum albumin (BSA) and 7 mM sodium azide) and manageable side effects,30 and thus we anticipated that this may then incubated with 50 ml of a polyclonal rabbit anti-Pseudo-