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Cancer Immunology, Immunotherapy

, Volume 57, Issue 3, pp 411–423 | Cite as

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells

  • Tina Müller
  • Christoph Uherek
  • Guitta Maki
  • Kai Uwe Chow
  • Annemarie Schimpf
  • Hans-Georg Klingemann
  • Torsten Tonn
  • Winfried S. Wels
Original Article

Abstract

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcγRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3ζ chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

Keywords

CD20 scFv antibody Natural killer cell Chimeric antigen receptor Adoptive therapy 

Notes

Acknowledgments

We thank Dr. Barbara Schnierle for providing pEFIRES-P vector, Dr. Annette Romanski for BV173 and NALM-6 cells, Dr. Byoung S. Kwon for anti-4-1BB antibody BBK-1, Daniela Bott for isolation of primary B and NK cells, Dr. Brigitte Rüster for help with microscopical analysis, Dipl. Ing. Nicola Krzossok for help with NK-92 cytotoxicity assays, Dr. Boris Brill, Sabrina Lehmen and Christiane Peter for help with animal experiments, and Dr. Markus Biburger for helpful suggestions. This work was supported in part by research grant 102386/10-2244 from Deutsche Krebshilfe. G. Maki was supported by grant CLL-63119, Section of Hematology, Rush University Medical Center.

Supplementary material

262_2007_383_MOESM1_ESM.pdf (59 kb)
Supplementary Fig. S1 Recombinant scFv(Leu-16) binds to CD20 expressing lymphoma cells. a For periplasmic expression of CD20-specific scFv(Leu-16) under control of the IPTGinducible tac promoter, cDNA fragments encoding heavy (VH) and light chain variable domains (VL) of monoclonal antibody Leu-16 were connected by a flexible linker sequence and fused to the ompA signal peptide (SP) in the bacterial expression vector pSW50. Synthetic FLAG- and Myc-tags are included at N- and C-termini of the gene product. b The presence of ErbB2-specific control protein scFv(FRP5) (lane 1) and CD20-specific scFv(Leu-16) (lane 2) in periplasmic extracts was confirmed by SDS-PAGE and immunoblot analysis with FLAG-tag specific Mab M2. c Binding of recombinant scFv molecules to CD20 expressing but ErbB2-negative Raji lymphoma cells (upper panel), and ErbB2 expressing but CD20-negative SKBR3 breast carcinoma cells was analyzed by flow cytometry with Myc-tag specific Mab 9E10 and FITC-conjugated secondary antibody. CD20-specific Mab L27 served as a control. (PDF 59 kb)
262_2007_383_MOESM2_ESM.pdf (9 kb)
Supplementary Fig. S2 Granzyme B activity is required for target cell killing by NK-92-scFv(Leu-16)-ζ cells. NK-92-scFv(Leu-16)-ζ cells were incubated with 100 μM of the serine protease inhibitor DCI (3,4-dichloroisocoumarin) (Roche, Mannheim, Germany) in X-VIVO 10 medium for 1 h at 37°C, before their cytotoxic activity towards CD20 expressing NIH3T3-CD20 cells was analyzed in a 3 h MTT cytotoxicity assay as described in the methods section (E/T ratio of 10:1). Untreated NK-92 and NK-92-scFv(Leu-16)-ζ cells, and NK-92-scFv(Leu-16)-ζ cells treated with DMSO served as controls. The relative number of viable target cells is expressed in % of NIH3T3-CD20 grown in the absence of NK cells (set to 100 %). Mean values of triplicate samples are shown. The standard deviation is indicated by error bars. At the concentration applied DCI was not toxic to NK-92 cells as evaluated by propidium iodide staining (data not shown). (PDF 8.85 kb)

Supplementary movie Selectivity and kinetics of target cell killing. NIH3T3-CD20 cells transduced with a retroviral vector encoding enhanced green fluorescent protein (eGFP) were mixed at a 1:1 ratio with parental NIH3T3 cells and grown overnight. Then NK-92-scFv(Leu-16)-ζ cells were added at an effector to target ratio of 1:1, microscopic images of a single field were taken at 1.5 min intervals for 6.4 h, and assembled into a QuickTime movie at 10 frames per second. At the beginning of the movie, a fluorescence microscopic image of eGFP- and CD20-positive NIH3T3-CD20(eGFP), and eGFP- and CD20-negative NIH3T3 cells before addition of NK cells is shown. Exemplary NIH3T3-CD20(eGFP) cells are indicated by white circles, exemplary parental NIH3T3 cells by black arrows. Selected images from this experiment are also shown in Fig. 5. (MOV 2.33 mb)

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Tina Müller
    • 1
  • Christoph Uherek
    • 1
  • Guitta Maki
    • 2
  • Kai Uwe Chow
    • 3
  • Annemarie Schimpf
    • 1
  • Hans-Georg Klingemann
    • 2
    • 4
  • Torsten Tonn
    • 5
  • Winfried S. Wels
    • 1
  1. 1.Chemotherapeutisches Forschungsinstitut Georg-Speyer-HausFrankfurt am MainGermany
  2. 2.Section of Bone Marrow Transplant and Cell TherapyRUSH Medical CollegeChicagoUSA
  3. 3.Department of Internal Medicine III, Hematology and OncologyUniversity Hospital FrankfurtFrankfurt am MainGermany
  4. 4.Division of Hematology/OncologyTufts-New England Medical CenterBostonUSA
  5. 5.Institute for Transfusion Medicine and Immunohematology RCBDSFrankfurt am MainGermany

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